Mechanistic Investigation of a Starch-Branching Enzyme Using Hydrodynamic Volume SEC Analysis

Hernandez, Javier. M., Gaborieau, Marianne., Castignolles, Patrice., Gidley, Mike, Myers, Alan. M. and Gilbert, Robert. G. (2008) Mechanistic Investigation of a Starch-Branching Enzyme Using Hydrodynamic Volume SEC Analysis. Biomacromolecules, 9 3: 954-965. doi:10.1021/bm701213p


Author Hernandez, Javier. M.
Gaborieau, Marianne.
Castignolles, Patrice.
Gidley, Mike
Myers, Alan. M.
Gilbert, Robert. G.
Title Mechanistic Investigation of a Starch-Branching Enzyme Using Hydrodynamic Volume SEC Analysis
Journal name Biomacromolecules   Check publisher's open access policy
ISSN 1525-7797
Publication date 2008-02-23
Year available 2008
Sub-type Article (original research)
DOI 10.1021/bm701213p
Open Access Status
Volume 9
Issue 3
Start page 954
End page 965
Total pages 12
Editor Albertson, Ann-Christine.
Place of publication United States
Publisher 2008 American Chemical Society
Language eng
Subject C1
030399 Macromolecular and Materials Chemistry not elsewhere classified
860199 Processed Food Products and Beverages (excl. Dairy Products) not elsewhere classified
Abstract Two linear alpha-(1,4)-D-glucans substrates, of degrees of polymerization DP similar to 150 and 6000, were exposed to maize starch-branching enzyme IIa (mSBEIIa) in vitro. The resulting branched alpha-glucans and their constituent chains (obtained by debranching) were analyzed by nuclear magnetic resonance (NMR) and size-exclusion chromatography (SEC). SEC data for the debranched species are presented as chain-length distributions, while those for branched species are presented as hydrodynamic volume distributions (HVDs), which is the most meaningful way to present such data (because SEC separates by size, not molar mass, and a sample of branched polymers with the same size can have a range of molar masses). A rigorous interpretation of the HVDs of the substrate and its branched product show that at least part of the branching is an interchain transfer mechanism in both the short- and long-chain substrate cases. A bimodal HVD of the in vitro branched alpha-glucan derived from the short-chain substrate was observed, and it is postulated that the divergence of the two populations is due to very small chains being unable to undergo branching. In the case of the in vitro branching of the long-chain substrate, the formation of maltohexaose during the reaction and the presence of a monomodal HVD were observed, suggesting a distinct mode of action of mSBEIIa on this substrate. Quantification of the branching level by NMR showed the branched glucans from both substrates had substantial amounts of branching (2.1-4.5%), ascribed to the intrinsic nature of the action of mSBEIIa on the two substrates. It is postulated that differences in the degrees of substrate association affect the pattern of branching catalyzed by the enzyme, and a putative active site structure is proposed based on the appearance of maltohexaose. The molar mass distribution of the constituent chains of the in vitro branched alpha-glucans obtained by isoamylase treatment reveals the transfer of chains of specific size and supports the supposition given in the literature that mSBEIIa is responsible for short-chain branching in amylopectin. It is suggested that hydrodynamic volume SEC analysis should be used as a tool for the mechanistic investigation of SBEs, allowing SEC data of in vitro branched alpha-glucans to be both comparable and quantitative.
Keyword Biochemistry & Molecular Biology
Chemistry, Organic
Polymer Science
Biochemistry & Molecular Biology
Chemistry
Polymer Science
BIOCHEMISTRY & MOLECULAR BIOLOGY
CHEMISTRY, ORGANIC
POLYMER SCIENCE
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

 
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Created: Thu, 16 Apr 2009, 00:59:28 EST by Emma Cushworth on behalf of School of Land, Crop and Food Sciences