UBC Theses and Dissertations
The role of adsorbed enzymes in determining the hydrolysis kinetics of pretreated lignocellulosic biomass Mok, Yiu Ki
The cost-effective production of sugars from biomass continues to remain challenging, partly due to the relatively high enzyme/protein loading required to effectively hydrolyze pretreated lignocellulosic substrates. Previous works have shown conflicting observations regarding the correlation between enzyme adsorption and the hydrolytic performance of an enzyme mixture. Unfortunately, it has proven difficult to accurately determine the roles of adsorbed enzymes during the hydrolysis of lignocellulosic substrates, in part because of the interference that protein determination methods encounter from the release of sugars and other biomass derived materials, the lack of a hydrolysis strategy for hydrolysis with only adsorbed enzymes and the use of “model” substrates in many studies. To better understand the role that adsorbed enzymes play in cellulose deconstruction, it is important that we are able to accurately quantify protein distribution and enzyme performance. Various protein quantification assays were initially assessed for their ability to accurately and reproducibly quantify protein/enzymes during typical biomass hydrolysis conditions. However, the ninhydrin assay, which was the most promising assay due to its specificity for protein and compatibility with most compounds derived from lignocellulosic samples, still suffered from the incompatibility with sugar degradation products, long hydrolysis times and potentially wide-ranging standard deviations. To overcome these limitations, an accurate and rapid modified ninhydrin assay was developed which employed a sodium borohydride treatment to eliminate sugar interference followed by acid hydrolysis at 130ºC, reducing the overall reaction time to 4 hours. Utilizing the modified ninhydrin assay, the role of adsorbed enzymes in determining the rate and extent of hydrolysis of several different pretreated biomass substrates was then assessed. Once the distribution of enzymes reached equilibrium, after 60 minutes, those enzymes that were adsorbed or free in solution were separated by centrifugation and subsequently assessed for their ability to hydrolyze various cellulosic substrates at different enzyme loadings. It was apparent that the adsorbed enzymes were critically important as the removal of those enzymes in solution resulted in no significant decrease in the rate and extent of hydrolysis. By using the adsorbed enzymes, enzyme loadings could be reduced by up to 53% while resulting in similar hydrolysis yields.
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