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

Abstract

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.

Popular Abstract

In this study, strong indications were found that the DNS method – a century old method for carbohydrate analysis – does not work as previously thought. The enzyme CGTase was also tested with different raw materials to gain a better understanding of how it works.
Until now, the DNS molecule has been though to only react with one specific part of certain sugars called reducing sugars. As the different kinds of reducing sugars all have one such part per molecule, this method has been used as an easy way of counting the total number of sugar molecules in a sample. In this study, however, more parts of the reducing sugar molecules reacted. This meant that larger molecules gave a higher signal. If this is general to all different ways of using the DNS method, then that would mean that the method has often been used incorrectly, and that conclusions from previous studies may be wrong.
In the second part of the study, it was investigated how the enzyme CGTase acts when it has different raw materials. The enzyme has the ability to catalyse four different reactions, which makes it possible to generate very different products. In general, the product profiles were similar, but a few observations were made. The enzyme produced longer products when it had circular sugars called α-cyclodextrins as raw material, and shorter products with short, straight sugar chains called maltodextrins. A general trend was found that, no matter what the raw material was, the end products followed a statistical distribution model that is sometimes seen in certain polymerisation reactions. It is important to understand the enzyme you are working with, as that makes it possible to give the enzyme the best possible opportunity to do its job.
The analysis of the enzyme’s products was done with HPAEC, which is a type of liquid chromatography designed specifically for carbohydrate analysis.

• author: Johansson, Matilda
• supervisor: Carl Grey; Stefan Ulvenlund
• organization: Biotechnology; Biotechnology (MSc)
• alternative title: Oligosackaridanalys - Utvärdering av DNS-metoden och specifikation av glukaner processade med CGTas
• course: KBTM05 20192
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: CGTase, DNS, Chromatography, HPAEC, HPAE, Carbohydrate, Carbohydrates, Glucans, Cyclodextrin glycosyltransferase, Biotechnology, Bioteknik, Flory, Enza, Enzyme
• language: English
• id: 9078969
• date added to LUP: 2022-05-04 09:29:39
• date last changed: 2022-05-04 09:29:39