Investigation of Carbohydrate Hydrolysing Enzymes in Applications for Characterisation and Synthesis of Carbohydrates

Cellulose and starch are non-toxic, biodegradable and modifiable polymers. These properties have made them increasingly important in industrial products e. g. in pharmaceutical applications and in technical dispersions. By chemical modification of these polymers the physical and chemical properties of the polymer can be adjusted to suit a certain area of application. The characteristics of modified celluloses and starches are to a great extent determined by the chemical nature of the substitutents and by the method used to achieve substitution. It is very important to be able to characterise the substitution pattern, both at the monomeric and at the polymeric level. Enhanced knowledge of the relationships between the substitution methods and the structure and properties of the polymer, can thus be used to create more cost efficient production methods. The focus of the thesis was on developing analytical techniques that can be used in the elucidation of substituent distribution of modified starches and celluloses by using carbohydrate hydrolysing enzymes as depolymerisation tools. The polymers are too large for most analytical instruments and thus they are made smaller selectively by enzymes. To know how substituents affect the enzymes ability to hydrolyse, the enzymes have to be characterised before they can be used as tools in polymer characterisation. Several analytical techniques (HPAEC-PAD, ESI-MS, SEC-RI) were used to analyse enzyme hydrolysis products. Also, apart from analysing the final products, intermediate products were analysed using µ-dialysis. This provided additional information on the enzymes? specificities. By immobilising the enzymes on a µ-chip reactor we also showed that enzymes are not only useful for cleaving, but also for building new carbohydrates by the covalent linking of a saccharide to an alcohol