UBC Theses and Dissertations
Activity-based proteomics profiling for identification and quantification of Trichoderma reesei cellulases Chen, Chi Fan
Cellulosic ethanol holds great promise as a renewable fuel to supplement gasoline. The complete conversion of cellulose involves multiple steps, one of which is the enzymatic degradation of cellulose to glucose. Trichoderma reesei (recently renamed as Hypocrea jecorina) secretes a large variety of cellulases that work synergistically in the hydrolysis of cellulose into glucose. However, enzymes tend to “die off” during biomass conversion, decreasing the efficiency of degradation, yet it is difficult to determine which enzyme in the mixture loses activity. Furthermore, significant diversity in the composition of plant cell walls requires optimization of enzyme mixture used for hydrolysis of each feedstock. In order to address the problems, tools to identify and quantitate the active enzyme species present in the enzyme mixtures are required, and would characterize the composition of the active enzymes in the hydrolysis mixtures. We proposed an activity-based protein profiling (ABPP) approach. It involves a set of chemical probes, each containing a cellulase-specific inactivating functionality to label the active enzymes in the hydrolysis mixture, and a reporter group to quantitate the modified enzymes using such reagents, the concentration of one specific enzyme could be determined based on the amount of labeled enzymes detected using the reporter group. A cellulasespecific inactivating functionality is either an affinity label or a mechanism-based inactivator incorporating a specificity-determining chemical group. To evaluate the potential inactivating functionalities, six cellobiose-based affinity labels based on reactive groups that have proved useful for other glycosidases, and two mechanism-based inactivators have been tested with the 4 principal T. reesei cellulases (provided by our collaborator) for their efficiencies. Seven of them inactivated at least one of the cellulases; among these, C-epoxypentyl cellobioside had the best overall performance. It had relatively good binding affinity and efficiency toward three cellulases, and it will be included in a second generation of ABPP probe. An enlarged inactivator library will be required to target other cellulases. Ultimately, at least one ABPP for each of the T. reesei cellulase will be generated and hopefully will prove useful to the biofuel industries.
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