title: Recombinant cyclodextrinase from Thermococcus kodakarensis KOD1: expression, purification, and enzymatic characterization.
abstract: 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 α-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 hydrolyze β-cyclodextrin (CD) but not α- or γ-CD, soluble starch, or pullulan. The final product from β-CD was glucose. The V max and K m values were 3.13 ± 0.47 U mg(-1) and 2.94 ± 0.16 mg mL(-1) for β-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.
authors: College of Plant Sciences, Jilin University, Changchun 130062, China.
authors: Ying
authors: 0000-0002-2042-6514
authors: Y
authors: Sun
authors: College of Plant Sciences, Jilin University, Changchun 130062, China.
authors: Xiaomin
authors: X
authors: Lv
authors: College of Plant Sciences, Jilin University, Changchun 130062, China.
authors: Zhengqun
authors: 0000-0002-6938-0705
authors: Z
authors: Li
authors: College of Plant Sciences, Jilin University, Changchun 130062, China.
authors: Jiaqiang
authors: 0000-0001-9068-8619
authors: J
authors: Wang
authors: College of Plant Sciences, Jilin University, Changchun 130062, China ; Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea.
authors: Baolei
authors: 0000-0001-6434-5604
authors: B
authors: Jia
authors: College of Plant Sciences, Jilin University, Changchun 130062, China.
authors: Jinliang
authors: 0000-0001-6605-1271
authors: J
authors: Liu
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004795:Enzyme Stability; D005947:Glucose / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics; D047392:beta-Cyclodextrins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase; D005947:Glucose