UBC Theses and Dissertations
Enzymatic synthesis and NMR investigation of cellulose I and II Ng, Emily Siu Pan
Cellulose is one of the most abundant naturally occurring polymers on earth. It has also long been a polymer of high interest. Surprisingly, the complete structure of cellulose still remains a mystery to this date. Therefore, this thesis will utilize the recent advances of fluoro-sugar chemistry and enzymology to produce celluloses and modified cellulose whose structures could be studied by high-resolution solid-state nuclear magnetic resonance spectroscopy. The in-vitro syntheses of cellulose I and II using wild-type enzymes (Trichoderma viride Onozuka R-10, Trichoderma viride, Trichoderma reesei, Aspergillus niger, Cellulomonas fimi) and mutant enzymes (E197A, E197G and E197S of endoglucanase I from Fusarium oxysporum) were investigated. In-vitro syntheses of cellulose I by varying the buffer systems in which the enzymatic polymerization reactions were carried out in was studied. Furthermore, the possibilities of investigating cellulose structure by incorporating fluorine atoms into its polymer structures were also explored. The substrates necessary for the enzymatic syntheses of cellulose, namely α-cellobiosyl fluoride (α-CBF), β-cellobiosyl fluoride (β-CBF), 6'-fluoro-6'-deoxy- α-cellobiosyl fluoride (6'F- α-CBF) and 6'-fluoro-6'-deoxy- β-cellobiosyl fluoride (6'F- β-CBF) were all synthesized. Then, various in-vitro enzymatic polymerizations of the above substrates using different enzymes were carried out in an array of different buffer systems. The buffer systems used in this study include an aqueous buffer system and 2:1, 5:1 and 7:1 ratios of acetonitrile to aqueous buffer systems. Interestingly, different buffer systems have dramatic effects on the amount of solid cellulose precipitates produced in each in-vitro polymerization regardless of the enzymes used. The resulting solid cellulose precipitates collected were then analysed using CP/MAS solid state NMR spectrometry. Unlike reports by Kobayashi and his group ref.¹⁻³, the in-vitro synthesis of cellulose I using 2:1 acetonitrile and aqueous buffer cannot be achieved on any of the enzymes tested. All cellulose samples produced were identified as cellulose II. Both 6'F- β-CBF and 6'F-α-CBF were successfully utilized by wild-type and mutant cellulases respectively as substrates and produced fluorinated cellulose polymers. Resulting fluorinated cellulose polymer were also analysed using CP/MAS solid state NMR spectrometry, proving that it is possible to use ¹⁹F CP/MAS NMR in combination with ¹³C and ¹H CP/MAS NMR to further the study of cellulose structures.
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