In Vitro Synthesis of Hyperbranched α-Glucans Using a Biomimetic Enzymatic Toolbox

Abstract

Glycogen biosynthesis requires the coordinated action of elongating and branching enzymes, of which the synergetic action is still not clearly understood. We have designed an experimental plan to develop and fully exploit a biomimetic system reproducing in vitro the activities involved in the formation of α(1,4) and α(1,6) glycosidic linkages during glycogen biosynthesis. This method is based on the use of two bacterial transglucosidases, the amylosucrase from Neisseria polysaccharea and the branching enzyme from Rhodothermus obamensis . The α-glucans synthesized from sucrose, a low cost agroresource, by the tandem action of the two enzymes, have been characterized by using complementary enzymatic, chromatographic, and imaging techniques. In a single step, linear and branched α-glucans were obtained, whose proportions, morphology, molar mass, and branching degree depended on both the initial sucrose concentration and the ratio between elongating and branching enzymes. In particular, spherical hyperbranched α-glucans with a controlled mean diameter (ranging from 10 to 150 nm), branching degree (from 10 to 13%), and weight-average molar mass (3.7 × 10(6) to 4.4 × 10(7) g.mol(-1)) were synthesized. Despite their structure, which is similar to that of natural glycogens, the mechanisms involved in their in vitro synthesis appeared to be different from those involved in the biosynthesis of native hyperbranched α-glucans.

Synthetic Polymers from Sugar-Based Monomers

“…Similarly, the synthesis of natural and non-natural polysaccharides (hyaluronan, chondroitin, and their derivatives belonging to glycosaminoglycans) was achieved by enzymatic polymerization using sugar oxazoline monomers and a glycoside hydrolase as catalyst. 228 Grimaud et al 229 designed a smart procedure to synthesize α-glucans from the sole substrate sucrose by the tandem action of two bacterial enzymes, i.e., the amylosucrose from Neisseria polysaccharea and the branching enzyme from Rhodothermus obamensis. The biomimetic system reproduced in vitro and in a single step the activities involved in the formation of α(1,4) and α(1,6) glycosidic linkages during glycogen biosynthesis.…”

Starch

“…Upon aging or drying, the amylose chains intertwined, resulting in the shrinking of the corona by the formation of crystallites and in a decrease of the particle diameter (Putaux et al 2006). Hyperbranched alpha-glucans have been recently produced by jointly using amylosucrase and a branching enzyme (Grimaud et al 2013) or phosphorylase and a branching enzyme (Ciric and Loos 2013). For a high ratio (around 340), the synthesized chains are long and reorganize in the form of a random organization of crystallites by a retrogradation process (Fig.…”

The Molecular Size Distribution of Glycogen and its Relevance to Diabetes

“…There have however been several encouraging studies using aqueous SEC to characterise synthetic and commercial glycogen. [9,11,34,35] There are several other advantages to using an aqueous system: water is cheaper, safer, easier to dispose of and more physiologically relevant than DMSO. The universal calibration assumption was used here.…”