The influence of α-1,4-glucan substrates on 4,6-α-d-glucanotransferase reaction dynamics during isomalto/malto-polysaccharide synthesis.

Starch-based isomalto/malto-polysaccharides (IMMPs) are soluble dietary fibres produced by the incubation of α-(1 → 4) linked glucans with the 4,6-α-glucanotransferase (GTFB) enzyme. In this study, we investigated the reaction dynamics of the GTFB enzyme by using isoamylase debranched starches as simplified linear substrates. Modification of α-glucans by GTFB was investigated over time and analysed with 1H NMR, HPSEC, HPAEC combined with glucose release measurements. We demonstrate that GTFB modification of linear substrates followed a substrate/acceptor model, in which α-(1 → 4) linked glucans DP ≥ 6 functioned as donor substrate, and α-(1 → 4) linked malto-oligomers DP < 6 functioned as acceptor. The presence of α-(1 → 4) linked malto-oligomers DP < 6 resulted in higher GTFB transferase activity, while their absence resulted in higher GTFB hydrolytic activity. The information obtained in this study provides a better insight into GTFB reaction dynamics and will be useful for α-glucan selection for the targeted synthesis of IMMPs in the future.

Enzymatic fingerprinting of isomalto/malto-polysaccharides.

In this study, we present an enzymatic fingerprinting method for the characterization of isomalto/malto-polysaccharides (IMMPs). IMMPs are produced by the modification of starch with the 4,6-α-glucanotransferase (GTFB) enzyme and consist of α-(1→4), α-(1→6) and α-(1→4,6) linked glucoses. Enzymes were used separately, simultaneously or in successive order to specifically degrade and/or reveal IMMP substructures. The enzymatic digests were subsequently analysed with HPSEC and HPAEC to reveal the chain length distribution (CLD) of different IMMP substructures. The presence of amylose in the substrate resulted in the formation of linear α-(1→6) linked glycosidic chains (13.5 kDa) in the former amylopectin fraction. The length of these chains indicates that GTFB transferase activity on amylopectin is more likely to elongate single amylopectin chains than to provide an even distribution. Enzymatic fingerprinting also revealed that the GTFB enzyme is capable of introducing large (20 kDa) linear α-(1→6) linked glycosidic chains in the α-glucan substrate.

Isomalto/malto-polysaccharide structure in relation to the structural properties of starch substrates.

Isomalto/malto-polysaccharides (IMMPs) are soluble dietary fibres produced by the enzymatic modification of starch with 4,6-α-glucanotransferase (GTFB). The structure, size, and linkage distribution of these IMMPs has remained largely unknown, since most structural information has been based on indirect measurements such as total α-(1→6) content, iodine staining and GTFB hydrolytic activity. This study provides a deeper understanding of IMMP structure in relation to its respective starch substrate, by combining preparative fractionation with linkage compositition analysis. IMMPs were produced from a variety of amylose-rich and amylose-free starches. The extent of modification was investigated per IMMP molecular weight (Mw)-fraction, distinguishing between linear α-(1→6) linkages introduced by GTFB and starch's native α-(1→4,6) branching points. It emerged that the amount of α-(1→6) linkages was consistently higher in IMMP low Mw-fractions and that GTFB activity was limited by native α-(1→4,6) linkages. The presence of amylose turned out to be a prerequisite for the incorporation of linear α-(1→6) linkages in amylopectin.