UBC Theses and Dissertations
How can we better predict the hydrolytic performance of commercial cellulase enzyme preparations on a range of biomass substrates? Mboowa, Drake
Predicting the hydrolytic potential of a “cellulase enzyme cocktail” on lignocellulosic substrates is an ongoing challenge, particularly as enzyme companies try to reduce the required enzyme loading to achieve rapid and complete cellulose hydrolysis. The filter paper assay (FPA) is still widely used to assess the hydrolytic potential of cellulase enzyme preparations, as the method is clearly documented and Whatman No.1 paper is a universally available and consistent substrate. However, characteristics of filter paper such as its high cellulose content and dried nature, as well as the short (1 hour) duration of the assay, etc., all compromise the ability of the FPA to predict how enzyme cocktails will hydrolyze lignocellulosic substrates. To assess the influence of factors such as drying and the presence of lignin and hemicellulose on the FPA, “model/paper sheet” substrates were prepared and substituted for filter paper. When a paper sheet prepared from never-dried pulp was used in the assay, it was apparent that drying was a major, negative influence. Using sheets prepared from pretreated substrates containing lignin and hemicellulose resulted in a 53% decrease in the measured filter paper activity, illustrating the detrimental effects of lignin and hemicellulose on cellulase activity. Therefore, several realistic substrates were prepared that were rich in either xylan, mannan and/or lignin. These substrates were used to assess the beneficial effects of substituting cellulases with accessory enzymes (e.g. xylanases, mannanases). In many cases up to 50% of the cellulases in the enzyme cocktail could be substituted with accessory enzymes to achieve hydrolysis yields >70%. However, when the substituted enzyme cocktail was assessed via the filter paper assay, the resulting activity decreased by up to 58%. It was apparent that the predictability of the FPA was highly dependent on the composition/characteristics of both the substrate and the enzyme cocktail. The results indicated a potentially more useful method to predict the effectiveness of a cellulase mixture on a given substrate may be to perform a longer-time hydrolysis of the actual biomass substrate while using the filter paper assay to provide an estimate of the initial protein/activity loading.
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