Biochemical characterization of a recombinant pullulanase from Thermococcus kodakarensis KOD1

Abstract

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. AbstractIn 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 5Á5-6Á0 and 100°C and exhibited good stability over a broad pH range (4-8). The V max and K m values were 118Á39 AE 1Á76 lmol mg À1 min À1 and 0Á37 AE 0Á02 mg ml À1 for pullulan and 53Á19 AE 11Á66 lmol mg À1 min À1 and 0Á36 AE 0Á05 mg ml À1 for starch.All these favourable enzymatic properties make it valuable in various industries.

Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis

“…In this study, we report a novel pullulan hydrolase of T. kodakarensis (TK-PUL) that was previously annotated (locus tag TK0977, GenBank accession no. BAD85166.1) and reported as a pullulanase type II (16,21). We prove here with convincing experimental results that TK-PUL is a pullulan hydrolase type III, rather than a type II.…”

Recombinant Cyclodextrinase fromThermococcus kodakarensisKOD1: Expression, Purification, and Enzymatic Characterization

“…Tk0977 can effectively hydrolyze starch to produce maltose and maltotriose. Tk1774 is an organic solvent-, detergent-, and thermostable amylopullulanase belonging to the GH57 family of proteins, and it only produces maltotriose [ 21 , 22 ]. These maltotriose products may be transported by an ABC-type maltodextrin transport system and further enter into the glycolytic pathway.…”

Extremophiles and biotechnology: current uses and prospects

“…Since the industrial processes involved in hydrolyzing starch require high temperatures (95°C for one step and 60°C for the other) and high pH, polyextremophilic (thermophilic and alkaliphilic) enzymes would be ideal. Currently, an α-amylase from Bacillus acidicola 57 , glucoamylases from Picrophilus 58 , and a pullulanase from Thermococcus kodakarensis 59 show great promise in replacing their mesophilic counterparts. However, amylases have also been isolated from halophiles, such as Halomonas meridian and Natronococcus amylolyticus , that could be useful in the process of producing high-fructose corn syrup, which is produced by hydrolyzing corn starch 43 .…”