An enzymatic tandem reaction is described in which the enzymes phosphorylase and Deinococcus geothermalis glycogen branching enzyme (Dg GBE) catalyze the synthesis of branched polyglucans from glucose‐1‐phosphate (G‐1‐P). Phosphorylase consumes G‐1‐P and polymerizes linear amylose while Dg GBE introduces branching points on the α‐(1 → 6) positions by reshuffling short oligosaccharides. The resulting branched polyglucans have an unusually high degree of branching of 11%.
Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of ␣1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (/␣) 7 -fold catalytic domain A with an inserted domain B between 2 and ␣5 and an ␣-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.
Densely packed polysaccharide brushes consisting of α-d-glucose residues were grafted from modified silicon substrates.
Potato phosphorylase was herein used to grow linear polysaccharide
chains from silicon tethered maltoheptaose oligosaccharides using
glucose-1-phosphate as donor substrate. The combined use of potato
phosphorylase and Deinococcus
geothermalis branching enzyme resulted in a hyperbranched brush coating as the
latter one redistributes short oligosaccharides from the α(1–4)-linked
position to the α (1–6)-linked position in the polysaccharide
brush. The obtained grafting density of the brushes was estimated
on 1.89 nm–2 while the thickness was measured with
ellipsometric techniques and determined to be between 12.2 and 20.2
nm.
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