Synthesis of Branched Polyglucans by the Tandem Action of Potato Phosphorylase and Deinococcus geothermalis Glycogen Branching Enzyme

Abstract

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%.

Synthesis of branched polysaccharides with tunable degree of branching

“…This is similar to the investigation of another group of enzymes reported in literature (Olsen et al, 1992). Furthermore, with the newly introduced enzyme, GPb, and a slight modification of pH values, addition of the enzyme activator and the reducing agent, branched polyglucans are synthesized with higher degree of branching than previously reported with potato phosphorylase (van der Vlist et al, 2008). This is possible due to the partial protonation of the Dg GBE, including the possibility of the protonation of the active site.…”

“…Dg GBE was produced as described in literature (Palomo, Kralj, van der Maarel, & Dijkhuizen, 2009). Potato phosphorylase was isolated and G-7 was synthesized as reported elsewhere (van der Vlist et al, 2008). 1 H NMR spectra were recorded on a Varian Inova 400 MHz spectrometer at 50 • C. 2,2-Dimethyl-2-silapentane-5-sulfonic acid (DSS) was used as an external standard.…”

“…Maltoheptaose was synthesized via ring opening of ␤-cyclodextrin according to literature (van der Vlist et al, 2008).…”

“…The tandem polymerization reaction was effectively performed with potato phosphorylase at a pH of 7.0 at 37 • C according to literature (van der Vlist et al, 2008). However, the optimum reaction conditions of GPb and Dg GBE are pH 6.9 to 7.4/38 • C (Brandt, Cafwlong, & Lee, 1975) and pH 8.0/34 • C, respectively.…”

“…For example, amylopectin analogue, hyper-branched amylose can be synthesized in vitro by combined action of phosphorylase (EC 2.4.1.1) and glycogen branching enzyme (GBE) (EC 2.4.1.18) (Fujii et al, 2003;Yanase, Takaha, & Kuriki, 2006). It was reported that branched polyglucans with the degree of branching of 11% can be synthesized by the tandem action of potato phosphorylase and Deinococcus geothermalis glycogen branching enzyme (Dg GBE) (van der Vlist et al, 2008).…”