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Design and Characterization of Multifunctional Xylanolytic Enzymes


Biodegradation of lignocellulose has been the focus of intense study because of its applicability in sustainable biotechnological processes. The use of enzymes for the processing of lignocellulosic material is a key step not only in the production of second generation bioethanol, but also presents an opportunity to reduce input and energy costs in the pulp and paper industry, in the food industry and in biorefinery. Enzymes represents a significant fraction of the costs associated with the saccharification of lignocellulosic material, and protein engineering strategies can be applied to improve the performance of these biocatalysts. The current project proposes to use rational and semi-rational methods of protein design to create multifunctional chimeric enzymes to act synergistically in the degradation of arabinoxylan, one of the most abundant polysaccharides in the cell wall of plants and a source of C5 and C6 sugars. The project involves a two-stage experimental strategy; 1) evaluate the products generated from the action of mixtures of xylyanalytic enzymes against the arabinoxylan substrate using LC-MS and HPAEC-PAD. This step will identify complementary enzymatic activities, as well as the combinations of enzymes that demonstrate synergistic activities, and 2) application of rational and semi-rational protein design methodologies to create multifunctional enzymes using a beta-prism scaffold protein designed ab initio in the LBBP, together with the characterization of the catalytic properties of the chimeric enzymes. (AU)