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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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Sequence and structure analysis of CDase-Tk. Cyclodextrinase sequences from T. kodakarensis KOD1 (CDase-Tk, Tk1770), Thermococcus sp. CL1 (CDase-Tc, YP_006424883.1), Thermococcus sp. B1001 (CDase-Tb, BAB18100.1), Pyrococcus furiosus (CDase-Pf, NP_579668.1), and Thermofilum pendens Hrk 5 (CDase-Tp, YP_920858.1) were aligned. The solid line indicates the four consensus regions conserved in the GH13 family. The asterisks show the positions of the three active sites. The conservation level of each residue is indicated by the height of the bars above each residue. The number at the ending of each line of amino acids indicates the number of the amino acid residues.
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fig1: Sequence and structure analysis of CDase-Tk. Cyclodextrinase sequences from T. kodakarensis KOD1 (CDase-Tk, Tk1770), Thermococcus sp. CL1 (CDase-Tc, YP_006424883.1), Thermococcus sp. B1001 (CDase-Tb, BAB18100.1), Pyrococcus furiosus (CDase-Pf, NP_579668.1), and Thermofilum pendens Hrk 5 (CDase-Tp, YP_920858.1) were aligned. The solid line indicates the four consensus regions conserved in the GH13 family. The asterisks show the positions of the three active sites. The conservation level of each residue is indicated by the height of the bars above each residue. The number at the ending of each line of amino acids indicates the number of the amino acid residues.

Mentions: CDase-Tk has 656 amino acids, a deduced MW of 76.4 kDa and a pI of 5.5 (http://web.expasy.org/compute_pi/). CDase-Tk does not have a predicted secretion signal. Compared with the CDase sequences available in GenBank, the CDase-Tk sequence is highly similar to that of corresponding genes, for example, genes from strain Thermococcus sp. CL1 (59%, YP_006424883.1), Thermococcus sp. B1001 (53%, BAB18100.1), Pyrococcus furiosus (56%, NP_579668.1), and Thermofilum pendens Hrk 5 (52%, YP_920858.1) (Figure 1). A UNIPROTKB Blastp search of the amino acid sequence of CDase-Tk suggested that residues 200–600 contain a signature typical of glycosyl hydrolase (GH) family 13, also known as the α-amylase family. The four conserved regions of all GH13 amylolytic enzymes were identified in the CDase-Tk sequence. Figure 1 shows an amino acid sequence alignment of some highly similar GH13 family proteins in which the amino acids Asp411, Glu437, and Asp502 of CDase-Tk correspond to the highly conserved catalytic residues in GH13 CDase.


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)

Sequence and structure analysis of CDase-Tk. Cyclodextrinase sequences from T. kodakarensis KOD1 (CDase-Tk, Tk1770), Thermococcus sp. CL1 (CDase-Tc, YP_006424883.1), Thermococcus sp. B1001 (CDase-Tb, BAB18100.1), Pyrococcus furiosus (CDase-Pf, NP_579668.1), and Thermofilum pendens Hrk 5 (CDase-Tp, YP_920858.1) were aligned. The solid line indicates the four consensus regions conserved in the GH13 family. The asterisks show the positions of the three active sites. The conservation level of each residue is indicated by the height of the bars above each residue. The number at the ending of each line of amino acids indicates the number of the amino acid residues.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Sequence and structure analysis of CDase-Tk. Cyclodextrinase sequences from T. kodakarensis KOD1 (CDase-Tk, Tk1770), Thermococcus sp. CL1 (CDase-Tc, YP_006424883.1), Thermococcus sp. B1001 (CDase-Tb, BAB18100.1), Pyrococcus furiosus (CDase-Pf, NP_579668.1), and Thermofilum pendens Hrk 5 (CDase-Tp, YP_920858.1) were aligned. The solid line indicates the four consensus regions conserved in the GH13 family. The asterisks show the positions of the three active sites. The conservation level of each residue is indicated by the height of the bars above each residue. The number at the ending of each line of amino acids indicates the number of the amino acid residues.
Mentions: CDase-Tk has 656 amino acids, a deduced MW of 76.4 kDa and a pI of 5.5 (http://web.expasy.org/compute_pi/). CDase-Tk does not have a predicted secretion signal. Compared with the CDase sequences available in GenBank, the CDase-Tk sequence is highly similar to that of corresponding genes, for example, genes from strain Thermococcus sp. CL1 (59%, YP_006424883.1), Thermococcus sp. B1001 (53%, BAB18100.1), Pyrococcus furiosus (56%, NP_579668.1), and Thermofilum pendens Hrk 5 (52%, YP_920858.1) (Figure 1). A UNIPROTKB Blastp search of the amino acid sequence of CDase-Tk suggested that residues 200–600 contain a signature typical of glycosyl hydrolase (GH) family 13, also known as the α-amylase family. The four conserved regions of all GH13 amylolytic enzymes were identified in the CDase-Tk sequence. Figure 1 shows an amino acid sequence alignment of some highly similar GH13 family proteins in which the amino acids Asp411, Glu437, and Asp502 of CDase-Tk correspond to the highly conserved catalytic residues in GH13 CDase.

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