Expression and characterization of fungal exocellulases

Although plant biomass is among the earth’s most abundant renewable resources, the high cost of converting it to usable sugars currently prevents it from being used as an effective replacement of petroleum as a feedstock for the production chemicals, power and fuels. One way of reducing costs is to improve the performance of the enzymes used to hydrolyze plant biomass. Bacteria and fungi that degrade plant biomass produce mixtures of enzymes, including endoglucanases, cellobiohydrolases and β-glucosidases, which work synergistically to hydrolyze the polymers in plant biomass to monomeric sugars. Biochemical parameters such as cell wall structure, pH and temperature can critically influence the rate and extent of plant biomass hydrolysis. Cellobiohydrolase (Cbh) types I and II release cellobiose by hydrolyzing cellulose polymers from their reducing and non-reducing ends. Here, I have made an attempt to establish benchmark cellulase hydrolysis assays using commercially available cellulase systems and the model substrate PASC. A library of fungal cellobiohydrolases were cloned and expressed using the filamentous fungus Aspergillus niger as the expression host. Activity assays were used to identify cellobiohydrolases that could be expressed as enzymatically active enzymes by A. niger. These enzymes were then subjected to biochemical characterization using enhancement studies with commercial cellulase mixtures. In addition, the biochemical properties of the characterized cellobiohydrolases were correlated with their primary sequence information. After initial screening based on levels of expression and enzyme activity, six cellobiohydrolases were selected as promising targets. Enhancement studies of commercial cellulase mixtures using four of these enzymes was done using two experimental designs. Although the properties of the different cellobiohydrolases were similar in many respects, there were significant differences in expression levels and relative activities, which may make some more attractive as candidates for cellulase system enhancement