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UBC Theses and Dissertations

Synthesis of cellulosic glycolipids using engineered enzymes Chambers, Martina Natasha


Cellulose, a linear polymer of D-glucose units connected by β-1,4 glycosidic bonds, adopts a highly-ordered crystalline structure in solution. In cellulose I, the dominant form of cellulose in nature, the polymeric chains are aligned in the same direction. Previous attempts to synthesize cellulose I in vitro have resulted in the synthesis of cellulose II, which has the thermodynamically favored anti-parallel orientation of chains. The synthesis of soluble fragments or defined surfaces of cellulose I would enable more detailed study of carbohydrate binding domains and other proteins that interact with cellulose in nature. The objective of this thesis is to prepare a crystalline surface of cellulose I in a controlled manner through the alignment of cellulolipids. A major focus of this thesis is the synthesis of cellulolipids with a cellohexaosyl head group. Cellohexaose is the shortest cello-oligosaccharide with cellulosic properties, but is consequently insoluble in aqueous solution. To improve the solubility of cellohexaose, the addition of a removable charged functionality was explored: either a terminal sialic acid or a phosphate group at the 6 position of the non-reducing terminal glucose. Abg2F6 glycosynthase from Agrobacterium sp. was used to synthesize β-1,4 linked cello-oligosaccharide fluorides from DP = 2 to DP = 4. These cello-oligosaccharides were modified with a removable charged functionality and utilized as donor substrates by CelB glycosynthase, a mutant of a β-1,4 endoglucanase from Caldicellulosiruptor saccharolyticus. Through the combination of glycosynthase enzymes and charged functionalities, a variety of soluble cellohexaosyl analogs were synthesized. Lyso-glycosphingolipids were prepared by transferring cello-oligosaccharyl fluorides to D-erythro-C18-sphingosine using EGCase glycosynthase. CelB glycosynthase used charged glycosyl fluoride donors to extend the lyso-glycosphingolipids, yielding soluble cellulolipids. The soluble cellulolipids were aligned along an aqueous:organic interface and the charged functionality was removed. Thus, a surface was prepared that appeared to interact with a carbohydrate binding module functionalized with a fluorescent tag. The soluble cellulolipids were successfully incorporated into a nanodisc, as shown through the incorporation of phosphorylated cellohexaosyl sphingosine. Cleavage of the phosphate using alkaline phosphatase yielded a nanodisc containing cellulolipids.

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