Abstract

A major bottleneck preventing the economically favourable production of biofuels from cellulose is the high cost of the cellulase enzymes used to convert cellulose into fermentable glucose units. One way to reduce the cost and increase the efficiency of cellulose hydrolysis is to develop new cellulase systems with enhanced cellulose hydrolysis rates and efficiencies. A better understanding of the relationship between cellulose structure and cellulose hydrolysis mechanisms will facilitate the development of cellulases with sufficiently enhanced hydrolysis rates and efficiencies. To achieve this, I am pursuing the following two research objectives: (i) express a library of evolutionarily diverse endoglucanases representing each of several different glycosyl hydrolase families and tertiary structures; and; (ii) perform detailed characterizations on the enzymes to gain insight into the relationship between cellulase structure and cellulase function. For these studies, I compared the hydrolysis capabilities of a library of novel endoglucanases against Trichoderma reesei (T. reesei) cellulase system endoglucanases using the model substrate phosphoric acid swollen cellulose (PASC), a substrate used in commercial ethanol production (pre-treated wheat straw), carboxymethylcellulose glucooligosaccharides and various hemicellulose components found in pretreated lignocellulose feedstocks. About 100 fungal genome sequences were downloaded from public databases. From these, 23 fungal species were selected based on their ability to hydrolyze vascular plant biomass and their evolutionary diversity. The 75 genes encoding endoglucanases selected from 23 fungal species were manually annotated, cloned and expressed using Aspergillus niger as the expression host. Congo Red indicator plate screening revealed that at least 37 of the endoglucanase genes expressed an active secreted endoglucanase (EG). Among the 37 EGs subjected to preliminary biochemical characterization, it was shown that the optimal pH and temperature of the 26 endoglucanases with the highest levels of activity ranged from pH 3.0 to pH 6.0 and from 40 ˚C to 70 ˚C. Based on the results of
this detailed screening, 17 endoglucanases were selected for further analyses that included: assessing their thermostability, assessing their ability to hydrolyze short oligosaccharides, determining their substrate specificity and assessing their ability to enhance the saccharification of cellulosic substrates by the T. reesei cellulase system and various derivatives thereof.