Recombinant Cyclodextrinase from Thermococcus kodakarensis KOD1: Expression, Purification, and Enzymatic Characterization

A gene encoding a cyclodextrinase from Thermococcus kodakarensis KOD1 (CDase-Tk) was identified and characterized. The gene encodes a protein of 656 amino acid residues with a molecular mass of 76.4 kDa harboring four conserved regions found in all members of the alpha-amylase family. A recombinant form of the enzyme was purified by ion-exchange chromatography, and its catalytic properties were examined. The enzyme was active in a broad range of pH conditions (pHs 4.0-10.0), with an optimal pH of 7.5 and a temperature optimum of 65 degrees C. The purified enzyme preferred to hydrolyze beta-cyclodextrin (CD) but not alpha-or gamma-CD, soluble starch, or pullulan. The final product from beta-CD was glucose. The V-max and K-m values were 3.13 +/- 0.47Umg(-1) and 2.94 +/- 0.16mgmL(-1) for beta-CD. The unique characteristics of CDase-Tk with a low catalytic temperature and substrate specificity are discussed, and the starch utilization pathway in a broad range of temperatures is also proposed.