Enzyme diagnostic criteria for polysaccharide purity: A model study using polymer reference standards

Some natural polysaccharides reported with complicated structures are possibly mixtures of simpler polymers. To address this concern, the concept of enzyme diagnostic criteria for polysaccharide purity has recently been proposed. Herein, a model study using polymer reference standards is conducted to test this conception. Reference standards of pullulan 50 kDa and dextran 50 kDa are mixed to make mixture model Ⅰ which represents a homopolysaccharide, and pullulan 10 kDa is mixed with polygalacturonic acid 10 kDa to make mixture model Ⅱ which represents heteropolysaccharide. However, both mixture models satisfactorily meet the current purity criteria, as assessed by High-Performance Gel Permeation Chromatography (HPGPC), monosaccharide composition analysis, methylation analysis, and 1D/2D-Nuclear Magnetic Resonance (NMR) analysis. Furthermore, conventional structure elucidation, based on the data of mixed structure features, yields complicated and incorrect structures as compared to the known structures of reference standards. By following the upgraded purity criteria, diagnostic enzymes are selected based on the same structure data, and their hydrolysis efficacy is subsequently validated. As a result, the changes in HPGPC peaks caused by diagnostic enzyme hydrolysis effectively reveal the presence of mixtures in both models. These findings clearly demonstrate the strength and efficiency of this new criteria for assessing polysaccharide purity.