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

[摘要] A gene encoding a cyclodextrinase fromThermococcus kodakarensisKOD1 (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 theα-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°C. The purified enzyme preferred to hydrolyzeβ-cyclodextrin (CD) but notα- orγ-CD, soluble starch, or pullulan. The final product fromβ-CD was glucose. TheVmaxandKmvalues were 3.13 ± 0.47 U mg−1and 2.94 ± 0.16 mg mL−1forβ-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.

[学科分类] 微生物学和免疫学