| Cellulose contains over half of all terrestrial organic carbon. An excellent way to tap this store of energy is to convert the cellulose into glucose and ferment the glucose to ethanol. This process yields a clean burning liquid fuel that can decrease our dependence on petroleum. Cellulases enzymatically hydrolyze cellulose to yield short glucose oligomers. In order to make cellulose to ethanol technology commercially viable the greatest potential for cost reduction is in improving the efficiency of enzymatic hydrolysis. One way to do this is to genetically engineer more efficient cellulases. In order to do this we must understand how these enzymes function.; The family 6 endocellulase E2 from the thermophilic bacterium Thermomonospora fusca was used to investigate the catalytic mechanism of cellulase action. This enzyme was chosen because its X-ray crystal structure is known to 1.18 A resolution. This study involved site directed mutagenesis of five different active site residues; Asp 79, Asp 117, Asp 156, Asp 265, and Tyr 73. These residues were selected because they are in or near the active site cleft, are conserved in all family 6 cellulases, and structural or molecular modeling data indicate that they may play a role in catalysis. The mutant proteins were expressed in Streptomyces lividans, purified and characterized.; The mutant enzymes were assayed for enzymatic activity on a variety of substrates. The activity of some mutant enzymes were tested as a function of pH. The binding affinities for small methylumbelliferyl labeled ligands as well as for cellotriose were determined. All mutant enzymes were shown to fold normally by circular dichroism spectra.; The data from these experiments showed that Asp 117 is the catalytic acid, Asp 156 functions to raise the pKa of Asp 117, and that Asp 265 and Tyr 73 are involved in binding. The role of Asp 79 is still unclear but it is tentatively proposed to be the catalytic base. |
