New Methods for Enzyme Hydrolysis, Analysis, and Characterization of Modified Cellulose

Sammanfattning: Cellulose can be chemically modified to produce derivatives with specific properties. Knowledge of correlations between the modification reaction, substituent distribution, origin of cellulose and properties are importaint in order to design processess so as to produce derivatives with the desired properties. This thesis describes research on newly developed analytical methods for the analysis of modified cellulose. Focus has been on developing new methods for the determination of the substituent distribution along the cellulose chain. This has been achieved by using specific enzyme hydrolysis of the modified cellulose. When the enzyme hydrolyses the modified cellulose, the substituents are considered as steric obstacles, making it more difficult for the enzyme to attack the cellulose chain. Thus, low substituted regions are generally hydrolysed to a higher extent than those that are highly substituted. By interpreting the formed oligomers after hydrolysis an attempt has been made to establish a picture of the substituent distribution of the intact polymer. A comparison to non-specific random acid hydrolysis has also been performed, making it possible to establish importaint information of the enzyme specificity to the substituents. The enzyme hydrolysis products have been analysed with several analytical techniques such as ESI-ITMS, MALDI-TOFMS, ESI-QqQMS, SEC-MALS/RI, HPAEC-PAD, and reducing ends. New ways of analysing the formed oligomers by selective marking of the formed oligomers, allowed for the analysis of how the substituents hinder the enzyme from gaining access to the modifed cellulose chain. Also, a novel method using MS was developed using internal standard addition that allowed for quantitative analysis of the hydrolysis products. Several new considerations when working with enzyme hydrolysed cellulose are suggested. Micro immobilised enzyme reactors have also been used to for the hydrolysis of the modified cellulose. These reactors made it possible to perform on-line hydrolysis of modified and unmodified cellulose connected to the techniques mentioned. The use of the reactors significantly reduced the amount of needed enzyme to perform the hydrolysis, reduced the amount of manual labour, and also significantly reduced the amount of time needed to perform the hydrolysis.