Oligosaccharide Analysis - Assessment of the DNS Method and Specification of Glucans Processed with CGTase

Sammanfattning: In this study, the reagent 3,5-dinitrosalicylic acid (DNS) and high-performance anion exchange chromatography (HPAEC) were used for carbohydrate characterisation. It was revealed that the DNS reaction is not exclusive to reducing ends as commonly thought, but rather that the reagent reacts with other parts of the carbohydrate molecules as well. HPAEC analysis was used to find how the product distribution is affected when cyclodextrin glycosyltransferase (CGTase) utilizes different substrates, and what effect an inert surfactant has on the outcome. The studies were performed in cooperation with Enza Biotech. The DNS method has been used for over a century to detect and quantify reducing ends of carbohydrates, as the reaction between a sugar and the DNS molecule is commonly thought to be exclusive to the reducing end aldehyde group. However, through analysing standard curves of maltooligosaccharides of different degrees of polymerization (DP), it was found that DNS molecules were reduced through reactions with other hydroxyl groups as well. Further studies are needed to find how specific this result is to the protocol used in this study. CGTase is a multifunctional enzyme with the unique ability to cyclize oligoglucans into cyclodextrins (CDs). In addition, it catalyses hydrolysis and two more transglycosylation reactions, all performed on α-1,4-glycosidic bonds. CGTase was used with four different substrate setups, namely α-CD (DP 6), maltodextrins of DEs (dextrose equivalents) 1 and 17, and DE 1 maltodextrin in combination with an inert surfactant. The reactions were run for 48 hours and speciation was performed by HPAEC-PAD analysis. The product profiles shared similar traits but differed in a few key areas. A higher average DP was observed within the linear fraction of the reaction where α CD was used as raw material. The reaction with the shorter maltodextrin stabilized fast and showed close to no net change, while the reaction with the longer maltodextrin showed changes in the product profile for the duration of the experiment and reached a lower DP. There was a strong indication that the presence of the inert surfactant led to the formation of inclusion complexes between the surfactant and the CDs. In general, all reactions finished with a major linear and a minor cyclic glycan fraction. The distribution of glucan species within the linear fractions of all reactions were distributed according to what resembled Flory statistics, a molecular weight distribution often observed in polymerization processes.