Understanding the mechanism of action of a processive endoglucanase and its application

Konar, Aditi (2026) Understanding the mechanism of action of a processive endoglucanase and its application. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Aditi Konar (20RS081)) 20RS081.pdf - Submitted Version Restricted to Repository staff only Download (7MB)

Abstract

Lignocellulosic biomass has emerged as a central feedstock for the global bioeconomy, offering sustainable routes to biofuels, bio-based chemicals, and high-value bioproducts. The efficient valorisation of this abundant resource fundamentally depends on its conversion into fermentable glucose, but its recalcitrant structure and limitations of existing cellulolytic systems hinder economically viable glucose production. To address this challenge, the first chapter of this thesis focuses on a processive endoglucanase (BlEG) that can directly produce glucose from lignocellulosic biomass, thereby reducing the dependence on a conventional multi-enzyme cellulase cocktail. Initial biochemical and biophysical characterization showed that the enzyme exhibits high catalytic activity and pronounced thermostability under relevant reaction conditions. The active site cleft was then mapped in detail, and the contribution of individual subsites to substrate binding and product formation was systematically examined through mutational analysis. In parallel, the role of the Carbohydrate binding module in substrate affinity, overall stability, and processivity was investigated. Finally, mutation of selected subsites revealed distinct changes in product profiles, allowing the identification of key structural features and amino acid residues that are critically associated with efficient glucose production from biomass. Following up from this, the second chapter addresses a major concern that limits cellulase performance, which is, feedback inhibition, where accumulation of the reaction product beyond a certain concentration suppresses enzymatic activity. Although docking tools can predict possible inhibitor-binding sites, experimental validation of these predicted allosteric sites is not straightforward. To overcome this, the chapter introduces a FRET-based assay specifically developed to verify the correct inhibitor-binding site. Two enzyme variants were assessed, one with the predicted allosteric site intact and another with the site disrupted, and a fluorophore was positioned near this region, while glucose was tagged with a donor fluorophore. Binding of glucose to the intact site resulted in a clear FRET signal, whereas the disrupted variant showed reduced FRET efficiency. The assay was also applied to β-glucosidases exhibiting glucose tolerance, stimulation, or inhibition, demonstrating its broader ability to resolve diverse glucose-enzyme interaction behaviours. Building on the objective of maximizing glucose production, the third chapter focuses on the rational design of a chimeric cellulase in which the endoglucanase BlEG, characterized in Chapter 1, is fused to a β-glucosidase to emulate natural enzymatic synergy. The construction of such chimeras inherently involves iterative optimization, as effective performance depends on the structural and functional compatibility between the two catalytic domains as well as the properties of the interconnecting linker, whether flexible or rigid. This approach enables the co-purification of two synergistic enzymes as a single fusion protein while also offering some improvements in overall biochemical performance. The fourth chapter integrates this glucose into a glucose-fed metabolic pathway to produce economically significant indole-based therapeutic precursors. For this purpose, the shikimate pathway of E. coli was engineered and selectively branched at the tryptophan-producing node to redirect flux toward indole-derived intermediates namely indole-pyruvic acid, indole-lactic acid, and indole-acrylic acid. In parallel, a bioassay system is being designed to allow real-time, intracellular monitoring of these metabolites within the E. coli host, facilitating rapid assessment of pathway performance and metabolic regulation. Together, these advances outline a circular bioeconomy framework in which agricultural biomass is directly converted into high-value therapeutic precursors through integrated enzyme engineering and microbial pathway design.

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