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Tantanarat, Krit, O'Neill, Ellis C., Rejzek, Martin, Field, Robert A. 
ORCID: https://orcid.org/0000-0001-8574-0275 and Limpaseni, Tipaporn
(2014)
Expression and characterization of 4-α-glucanotransferase genes from Manihot esculenta Crantz and Arabidopsis thaliana and their use for the production of cycloamyloses.
Process Biochemistry, 49 (1).
pp. 84-89.
ISSN 1359-5113
Abstract
4-α-Glucanotransferase or disproportionating enzyme (D-enzyme, DPE) catalyzes the α-1.4 glycosyl transfer between oligosaccharides. Type I D-enzyme (DPE1) can transfer maltosyl unit from one 1.4-α-d-glucan to an acceptor mono- or oligo-saccharide, which reflects the physiological role of DPE1 in plant starch metabolism. In this study, the genes encoding DPE1 from Arabidopsis thaliana (AtDPE1) and Manihot esculenta Crantz cultivar KU50 (MeDPE1) were cloned and expressed in Escherichia coli and purified to homogeneity. MeDPE1 encoded 585 amino acid residues, including a 56 residue signal peptide, while AtDPE1 encoded 576 amino acid residues with a 45 residue signal peptide. The molecular mass of both mature enzymes, estimated from deduced amino acid sequence, were the same at 59.4 kDa, with a pI of 5.13. The predicted structures of both enzymes showed the conserved 250's loop and three catalytic amino acid residues, characteristics of disproportionating enzymes in the GH77 glycoside hydrolase family. Biochemical characterization showed that both purified recombinant enzymes were homodimers in solution, with similar optimum pH and temperature for disproportionating activity at pH 6-8 and 37 C. Using potato amylose as a substrate, AtDPE1 can produce cycloamyloses in the range 16-50 glucose residues, while products from the action of MeDPE1 on the same substrate were in a wider range of 16 to DP > 60. These recombinant enzymes are useful tools for elucidation of their functional roles in starch metabolism and for applications in the starch industry.
| Item Type: | Article |
|---|---|
| Additional Information: | Funding Information: This work was performed while KT was a recipient of the Royal Golden Jubilee PhD Fellowship from the Thailand Research Fund and postdoctoral fellowships from Chulalongkorn University Graduate School. Research supports was provided by the National Research Council of Thailand in cooperation with the National Science and Technology Development Agency and IIAC under Chulalongkorn University Centenary Academic Development Project, and the UK BBSRC Institute Strategic Programme Grant on Understanding and Exploiting Metabolism (MET) [ BB/J004561/1 ] and the John Innes Foundation . |
| Uncontrolled Keywords: | arabidopsis thaliana,cycloamylose,d-enzyme,gh77 family,manihot esculenta crantz,bioengineering,biochemistry,applied microbiology and biotechnology ,/dk/atira/pure/subjectarea/asjc/1500/1502 |
| Faculty \ School: | Faculty of Science > School of Chemistry, Pharmacy and Pharmacology |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 04 Sep 2024 13:35 |
| Last Modified: | 25 Sep 2024 18:06 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/96540 |
| DOI: | 10.1016/j.procbio.2013.10.009 |
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