A New Method for in vitro Glycogen Synthesis, and the Structure and Properties of the Synthesized Glycogen

Abstract

A new enzymatic process for glycogen production was developed. In this process, short-chain amylose is used as the substrate for branching enzymes (BE, EC 2.4.1.18). The molecular weight of the enzymatically synthesized glycogen (ESG) depends on the type of BE used and the molecular weight and the concentration of the substrate amylose. Although a plant BE (kidney bean) and Bacillus cereus BE could not synthesize high molecular weight glucan, BEs from 6 other bacterial sources produced ESG. The BE from Aquifex aeolicus was the most suitable for the production of glycogen with a weight-average molecular weight (M_w) of 3000 kDa to 30,000 kDa. Furthermore, the addition of amylomaltase (AM, EC 2.4.1.25) significantly enhanced the efficiency of this process, and the yield of ESG reached approximately 65%. Typical preparations of ESG obtained by this method were subjected to structural analyses. The average chain length, interior chain length, and exterior chain length of the ESGs were 8.2-11.6, 2.0-3.3 and 4.2-7.6, respectively. Transmission electron microscopy showed that the ESG molecules formed spherical particles. Viscometric analyses also supported a spherical nature for the product. Unlike starch, the ESGs were barely degraded by pullulanase. Solutions of ESG were opalescent (milky-white and slightly bluish), and gave a reddish-brown color on the addition of iodine. These analyses revealed that ESG shares similar molecular shapes and solution properties with natural source glycogen (NSG).