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Expression and characterization of 4-α-glucanotransferase genes from Manihot esculenta Crantz and Arabidopsis thaliana and their use for the production of cycloamyloses

Paper ID Volume ID Publish Year Pages File Format Full-Text
34827 45051 2014 6 PDF Available
Title
Expression and characterization of 4-α-glucanotransferase genes from Manihot esculenta Crantz and Arabidopsis thaliana and their use for the production of cycloamyloses
Abstract

•Disproportionating enzyme from Manihot esculenta Crantz and Arabidopsis thaliana were cloned, over-expressed in E. coli.•The hypothetical structures of the two enzymes were elucidated.•The DPE1 of cassava Manihot esculenta Crantz was biochemically characterized for the first time.•Both DPE1 showed distinct ability to form large ring cycloamyloses with minimum size at 16 glucose residues.

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.

Keywords
D-enzyme; GH77 family; Manihot esculenta Crantz; Arabidopsis thaliana; Cycloamylose
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Expression and characterization of 4-α-glucanotransferase genes from Manihot esculenta Crantz and Arabidopsis thaliana and their use for the production of cycloamyloses
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 49, Issue 1, January 2014, Pages 84–89
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us