Deciphering Cellulase Mechanisms: A Combined Computational and Experimental Approach to Understand Sugar, Ionic Liquid, and Thermal Tolerance

Sahu, Sneha (2026) Deciphering Cellulase Mechanisms: A Combined Computational and Experimental Approach to Understand Sugar, Ionic Liquid, and Thermal Tolerance. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Sneha Sahu (19IP012)) 19IP012.pdf - Submitted Version Restricted to Repository staff only Download (11MB)

Abstract

The increasing demand for renewable and sustainable energy has intensified interest in producing biofuels from lignocellulosic biomass. Cellulases play a central role in this process by hydrolyzing cellulose into fermentable sugars. However, their industrial application is constrained by low catalytic efficiency, product inhibition, low tolerance to pretreatment methods such as ionic liquids (IL), and low thermostability. Overcoming these challenges is essential for economically viable biofuel production. While enzyme engineering has yielded important advances, it is often time and resource-intensive. Integrating molecular dynamics (MD) simulations with experimental approaches offers an efficient strategy to uncover molecular determinants of enzyme function, stability, and inhibition, enabling rational enzyme design. This thesis focuses on β-glucosidase (BG), the enzyme responsible for the final step in cellulose hydrolysis, converting cellobiose into glucose. Although its catalytic mechanism is conserved across mesophilic to hyperthermophilic environments, the molecular basis of thermal adaptation remains poorly understood. I first characterized the temperature-dependent activity of the mesophilic β-glucosidase BglB from Paenibacillus polymyxa and subsequently extended the analysis to β-glucosidases from multiple glycoside hydrolase families spanning mesophiles to hyperthermophiles. By integrating MD simulations with a curated dataset derived from sequence analysis and phylogeny, I establish connections among sequence variation, structural dynamics, and functional adaptation across diverse cellulases. The thesis further examines product-mediated inhibition, showing that glucose modulates catalytic activity through interactions with surface secondary binding sites and long-range structural communication networks. Simulation-guided mutagenesis and experimental validation probed these mechanistic insights. Finally, the framework was extended to a cellobiohydrolase to investigate stability in ionic liquid, identifying structural determinants of IL tolerance and catalytic robustness. Overall, this work demonstrates that integrated computational and experimental approaches provide a powerful platform for understanding cellulase mechanisms and guiding the engineering of enzymes with improved industrial performance.

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