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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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Thin layer chromatography (TLC) of hydrolysis products from β-CD generated by CDase-Tk. Lane 1: 1% β-CD alone, Lanes 2 to 4: CDase-Tk which was reacted with substrates at 1% concentration at 65°C for 10, 30, or 60 min. Std indicates the oligosaccharide standard containing 1% glucose, maltotriose, maltopentaose, and maltoheptaose.
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fig5: Thin layer chromatography (TLC) of hydrolysis products from β-CD generated by CDase-Tk. Lane 1: 1% β-CD alone, Lanes 2 to 4: CDase-Tk which was reacted with substrates at 1% concentration at 65°C for 10, 30, or 60 min. Std indicates the oligosaccharide standard containing 1% glucose, maltotriose, maltopentaose, and maltoheptaose.

Mentions: The kinetics of recombinant CDase-Tk were analyzed using β-CD as a substrate by varying its concentration. The reaction was performed in a Tris-HCl buffer (pH 7.5) at 65°C with β-CD concentrations ranging from 1 to 10 mg mL−1. The Michaelis–Menten equation was used to calculate the kinetic parameters (Figure 4). CDase-Tk catalyzed β-CD with Km = 3.13 ± 0.47 mg mL−1 and Vmax⁡ = 2.94 ± 0.16 U mg−1. TLC results demonstrated that the action of CDase-Tk results in the formation of glucose when using β-CD as a substrate (Figure 5). Other CDases show a broad range of substrates and products. For example CDase-Pf, a cyclodextrinase from GH13, possesses characteristics of both α-amylase and cyclodextrin-hydrolyzing enzyme. Similar to typical α-amylases, CDase-Pf hydrolyzes maltooligosaccharides and starch to mainly produce maltotriose and maltotetraose. However, this enzyme could also attack and degrade pullulan and β-CD [15] (Table 1).


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)

Thin layer chromatography (TLC) of hydrolysis products from β-CD generated by CDase-Tk. Lane 1: 1% β-CD alone, Lanes 2 to 4: CDase-Tk which was reacted with substrates at 1% concentration at 65°C for 10, 30, or 60 min. Std indicates the oligosaccharide standard containing 1% glucose, maltotriose, maltopentaose, and maltoheptaose.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Thin layer chromatography (TLC) of hydrolysis products from β-CD generated by CDase-Tk. Lane 1: 1% β-CD alone, Lanes 2 to 4: CDase-Tk which was reacted with substrates at 1% concentration at 65°C for 10, 30, or 60 min. Std indicates the oligosaccharide standard containing 1% glucose, maltotriose, maltopentaose, and maltoheptaose.
Mentions: The kinetics of recombinant CDase-Tk were analyzed using β-CD as a substrate by varying its concentration. The reaction was performed in a Tris-HCl buffer (pH 7.5) at 65°C with β-CD concentrations ranging from 1 to 10 mg mL−1. The Michaelis–Menten equation was used to calculate the kinetic parameters (Figure 4). CDase-Tk catalyzed β-CD with Km = 3.13 ± 0.47 mg mL−1 and Vmax⁡ = 2.94 ± 0.16 U mg−1. TLC results demonstrated that the action of CDase-Tk results in the formation of glucose when using β-CD as a substrate (Figure 5). Other CDases show a broad range of substrates and products. For example CDase-Pf, a cyclodextrinase from GH13, possesses characteristics of both α-amylase and cyclodextrin-hydrolyzing enzyme. Similar to typical α-amylases, CDase-Pf hydrolyzes maltooligosaccharides and starch to mainly produce maltotriose and maltotetraose. However, this enzyme could also attack and degrade pullulan and β-CD [15] (Table 1).

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