Initial velocity analyses of milling effects on rates of enzyme-catalyzed saccharification of native wheat straw

Graduation date: 2011There is considerable interest in developing environmentally friendly processes for the economical conversion of biomass plant material to biofuels, bioproducts and biomaterials. One approach to such a conversion process is to make use of enzymes for the conversion of plant polysaccharides to simple sugars; the simple sugars could then be chemically or biologically converted to any number of products. This thesis aims to provide information that will aid the development of such processes. The overall objective of the research is to test the validity of applying initial velocity kinetics to understand the parameters that dictate rates of biomass cellulose saccharification as catalyzed by a commercial cellulase preparation. The substrate chosen for the study was native wheat straw. The commercial enzyme preparation used in this study is a complex mixture containing multiple cellulolytic enzymes as well as accessory enzymes. The accessory enzymes, although not directly catalyzing reactions involving cellulose, may aid cellulose saccharification due to their activity on plant cell wall structural components that are associated with cellulose. The experimental variable chosen for study via initial velocity experiments was substrate particle size; specifically, how milling wheat straw to a smaller particle size may impact its rate of saccharification. It is expected that particle size, as related to surface area, will play an important role in governing the rate of this heterogeneous reaction. Considerable effort was put into determining feasible conditions\ud for initial velocity measurements, including the avoidance of non-cellulose glucose generated during saccharification. Time course experiments covering 24-hour reactions were included along with the initial velocity experiments. Time course experiments demonstrated that wheat straw preparations milled to pass sieves ranging in exclusion limits from 4 mm to 0.25 mm differed in rates of reaction as predicted, the smaller the particle size the greater the extent of reaction. However, substrates differing 16-fold with respect to their sieving exclusion limit differed less than 2-fold in their rates of saccharification. Initial velocity experiments focused on a comparison of a substrate milled to pass a 1 mm screen and one milled to pass a 0.25 mm screen. The initial rate of saccharification of the < 0.25 mm substrate was found to be approximately 15% greater than that of the < 1 mm substrate. This increase in rate approximates that to be expected based on theoretical calculations for changes in surface area. The data generated in the analysis of these substrates was consistent with that supporting the application of initial velocity methods. The combined results demonstrate the successful use of the initial velocity approach to the study of this type of heterogeneous (soluble enzyme/insoluble substrate) system. A further insight garnered from the initial velocity approach was that this system, i.e. a multi-enzyme complex catalyzing the saccharification of native wheat straw, does not obey simple saturation kinetics. This behavior has been tentatively attributed to the presence of both cellulolytic enzymes and accessory enzymes in the commercial enzyme preparation, and that these two classes of enzymes differ with respect to the enzyme load necessary for substrate saturation