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theses

Conversion of inedible lignocellulosic plant biomass, composed primarily of carbohydrate polymers like cellulose, into biofuels can quench the ever-growing societal demands for sustainable and renewable forms of energy. Naturally occurring microbial enzymes called cellulases, that are extracellularly secreted by industrial workhorse fungi such as Trichoderma reesei, can aid in the deconstruction of lignocellulosic feedstock to soluble sugars (for further upgrading to fuels) using benevolent enzymatic hydrolysis based processes unlike high thermochemical severity based acid-catalyzed processes. The most abundant cellulase expressed by T. reesei has a two-domain structure consisting of a Carbohydrate Binding Module (CBM) and a Catalytic Domain (CD) linked by a glycosylated linker peptide that catalyzes cellulose hydrolysis to cellobiose. This CBM was classified as the first cellulose – binding protein family (or CBM1) of its type since it was the first CBM to be discovered and belongs to family 1 (or CBM1). CBMs are auxiliary carbohydrate-active enzyme (CAZyme) protein domains that aid in the binding of enzymes to carbohydrate substrates as well as improve the activity of the catalytic domain through mechanisms that are still far from being fully understood. Cellulose exists naturally in plant cell walls as self-assembled microfibrils and has a defined crystalline allomorphic structure called cellulose-I. The tight packing of cellulose fibrils lowers accessibility to cellulases, thus severely limiting the rate of cellulose enzymatic hydrolysis. Pretreatment of native cellulose with anhydrous liquid ammonia can restructure the hydrogen bonds network to form an unnatural allomorph called cellulose-III that is more easily digestible by some families of cellulolytic enzymes. Wild-type family 1 CBMs and their engineered mutants’ characterization was the main focus of this study. These proteins were expressed fused on the C-terminus to a Green Fluorescent Protein (GFP), purified using a two-step purification process and their adsorption to cellulose-I and cellulose-III were systematically studied. Effect of physical parameters such as pH and ionic strength on CBM binding to both allomorphs of cellulose was also studied. This study helps understand the role of conserved amino residues on the flat binding face of CBM1 that impact cellulase binding to native and unnatural cellulose allomorphs. This would ultimately impact cost-effective conversion of cellulosic biomass to biofuels or biochemicals using CAZymes in an industrial biorefinery.

ETD_8321

M.S.

Includes bibliographical references

by Vibha Narayanan

doi:10.7282/T3ZG6WCK

ETD graduate

The author owns the copyright to this work.