Controlling the Fine Structure of Glycogen-like Glucan by Rational Enzymatic Synthesis

Abstract

Glycogen-like glucan (GnG), a hyperbranched glucose polymer, has been receiving increasing attention to generate synthetic polymers and nanoparticles. Importantly, different branching patterns strongly influence the functionality of GnG. To uncover ways of obtaining different GnG branching patterns, a series of GnG with radius from 22.03 to 27.06 nm were synthesized using sucrose phosphorylase, α-glucan phosphorylase (GP), and branching enzyme (BE). Adjusting the relative activity ratio of GP and BE (GP/BE) made the molecular weight (MW) distribution of intermediate GnG products follow two different paths. At a low GP/BE, the GnG developed from "small to large" during the synthetic process, with the MW increasing from 6.15 × 10 6 to 1.21 × 10 7 g/mol, and possessed a compact structure. By contrast, a high GP/BE caused the "large to small" model, with the MW reduction of GnG from 1.62 × 10 7 to 1.21 × 10 7 g/mol, and created a loose external structure. The higher GP activity promoted the elongation of external chains and restrained chain transfer by the BE to the inner zone of GnG, which would modulate the loose−tight structure of GnG. These findings provide new useful insights into the construction of structurally well-defined nanoparticles.

Effect of Starch Primers on the Fine Structure of Enzymatically Synthesized Glycogen-like Glucan

“…The glgB gene was cloned for expression and production of BE from Aquifex aeolicus VF5 (GenBank accession number AAC06895.1) using the pET-28­(+) vector as described by Takata et al The details of protein purification are given in the Supporting Information. Recombinant SP from Streptococcus mutans (GenBank accession number AAN58596.1) contained eight mutations (T47S, S62P, Y77H, V128L, K140M, Q144R, N155S, and D249G), and the mutated gtfA gene was inserted into NdelI/BamHI-digested pET-15b as described by Liu et al and Fujii et al The glgP gene from A. aeolicus VF5 (GenBank accession number AAC06896.1) was cloned for expression and production of GP and inserted into the EcoR1-Nde1 sites of the pET-28b vector as described by Liu et al and Bhuiyan et al All recombined plasmids were synthesized by BGI Genomics Co., Ltd. (Beijing, China) and transformed into Escherichia coli BL21­(DE3)*.…”

“…The GP activity was measured by the amount of released Pi at 55 °C using molybdate reagent as described by Liu et al and Kadokawa . One unit of GP activity was defined as the amount of the enzyme that produced 1 μmol Pi per minute under the assay conditions.…”

“…The GnG synthesis was modified from the method described by Liu et al and Ryoyama et al . The native and BE-pretreated starches (50 mg) were dispersed in 20 mL of phosphate buffer (160 mM KH 2 PO 4 , 240 mM Na 2 HPO 4 , pH 7) and gelatinized as above.…”

“…Branching Enzyme. The BE activity was assayed by the iodine staining method at 55 °C as described by Liu et al 25 and Takata et al 24 One unit of BE activity was defined as the amount of enzyme giving rise to a 1% decrease per minute in absorbance at 660 nm of the amylose−iodine complex under the assay conditions.…”

“…α-Glucan Phosphorylase. The GP activity was measured by the amount of released Pi at 55 °C using molybdate reagent as described by Liu et al 25 and Kadokawa. 29 One unit of GP activity was defined as the amount of the enzyme that produced 1 μmol Pi per minute under the assay conditions.…”