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Recombinant cyclodextrinase from Thermococcus kodakarensis KOD1: expression, purification, and enzymatic characterization.

Sun Y, Lv X, Li Z, Wang J, Jia B, Liu J - Archaea (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: College of Plant Sciences, Jilin University, Changchun 130062, China.

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.

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Proposed model for the degradation of starch in T. kodakarensis KOD1.
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fig6: Proposed model for the degradation of starch in T. kodakarensis KOD1.

Mentions: Previously, we reported that two extracellular pullulanases in T. kodakarensis KOD1 (Tk0977 and Tk1774) can hydrolyze pullulan and starch to an oligosaccharide with optimal temperatures above 100°C. Tk0977 is a protein of 765 amino acids with a putative 22-residue signal peptide. This protein has four consensus motifs and a catalytic triad of the GH13 family in the deduced amino acid sequence. 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. In this study, a pathway comprising a CGTase and CDase in T. kodakarensis KOD1 catalyzed the extracellular formation of β-CD from starch, and its subsequent intracellular degradation was reported. The CGTase-CDase pathway showed optimal catalytic characteristics at a lower temperature. Based on these observations, we propose that the four enzymes (Tk0977, Tk1770, Tk1774, and Tk2172) participate in the process of starch utilization synergistically with a broad temperature range to provide glucose for cell metabolism (Figure 6).


Recombinant cyclodextrinase from Thermococcus kodakarensis KOD1: expression, purification, and enzymatic characterization.

Sun Y, Lv X, Li Z, Wang J, Jia B, Liu J - Archaea (2015)

Proposed model for the degradation of starch in T. kodakarensis KOD1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321091&req=5

fig6: Proposed model for the degradation of starch in T. kodakarensis KOD1.
Mentions: Previously, we reported that two extracellular pullulanases in T. kodakarensis KOD1 (Tk0977 and Tk1774) can hydrolyze pullulan and starch to an oligosaccharide with optimal temperatures above 100°C. Tk0977 is a protein of 765 amino acids with a putative 22-residue signal peptide. This protein has four consensus motifs and a catalytic triad of the GH13 family in the deduced amino acid sequence. 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. In this study, a pathway comprising a CGTase and CDase in T. kodakarensis KOD1 catalyzed the extracellular formation of β-CD from starch, and its subsequent intracellular degradation was reported. The CGTase-CDase pathway showed optimal catalytic characteristics at a lower temperature. Based on these observations, we propose that the four enzymes (Tk0977, Tk1770, Tk1774, and Tk2172) participate in the process of starch utilization synergistically with a broad temperature range to provide glucose for cell metabolism (Figure 6).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: College of Plant Sciences, Jilin University, Changchun 130062, China.

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.

Show MeSH
Related in: MedlinePlus