Graduate Thesis Or Dissertation


Using Chemical Synthesis to Investigate Protein Glycosylation: O-Mannosylation Site Specifically Modulates the Stability and Cellulose Binding Affinity of Family 1 Carbohydrate Binding Modules Public Deposited
  • Lignocellulosic biomass is a massive, but largely unexploited potential source of biofuel. The underutilization of this resource stems largely from the fact that cellulose is difficult to digest into smaller, useable sugar units. Natural lignocellulosic biomass is primarily degraded by fungi, which use Family 1 carbohydrate-binding modules (CBMs) to target cellulose for degradation. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation of the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase (TrCel7A) are investigated at three glycosylation sites, Thr-1, Ser-3 and Ser-14. The work was made possible by the development of a convenient one-pot synthetic procedure for glycosylated Family 1 CBMs. A library of 20 CBM glycoforms was synthesized with mono-, di-, or tri-saccharides at each glycosylation site. The binding affinity, proteolytic stability, and thermostability of each synthetic glycoform was systematically studied. The results show that even though CBM mannosylation does not induce significant changes to the protein’s secondary structure, it can increase the thermolysin cleavage resistance up to 50-fold. Fungi are known to excrete several proteases along with CBM-bearing cellulases, so improved proteolytic stability may improve cellulose digestion efficiency by reducing CBM degradation. O-mannosylation was also shown to increases the thermostability of CBM glycoforms up to 16°C, and a mannose disaccharide at Ser3 has the largest themostabilizing effect. Thermostability is an important property of industrial enzymes because bioreactors are often operated at elevated temperatures. In the binding affinity tests, the glycoforms with small glycans at each site displayed the highest binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of the three positions had the highest binding affinity; a 7.4 fold increase compared to the unglycosyalted CBM. High CBM binding affinity has been linked to increased cellulose digestion rate by fungal cellulases, so these results may have important implications in biofuels production. This study demonstrated how chemical synthesis can be used to systematically study glycosylation and lead to the identification of two CBM glycoforms with particularly desirable stability and binding properties.
Date Issued
  • 2014
Academic Affiliation
Committee Member
Degree Grantor
Commencement Year
Last Modified
  • 2019-11-18
Resource Type
Rights Statement