Studying the role of FN3 domain and lignin inhibition in a thermophilic β-glucosidase

Panda, Swarnendu (2025) Studying the role of FN3 domain and lignin inhibition in a thermophilic β-glucosidase. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Swarnendu Panda (20MS023)) 20MS023_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (2MB)

Abstract

Efficient enzymatic degradation of lignocellulosic biomass is critical for sustainable biofuel production, yet lignin-mediated enzyme inhibition and poor substrate targeting remain major bottlenecks. This work addresses these challenges by elucidating FN3’s cellulose-binding mechanism, quantifying lignin’s inhibitory effects on β-glucosidase activity, and validating FN3’s structural stability-insights essential for engineering robust biocatalysts in biomass conversion. First, fluorescence microscopy revealed that GFP-tagged FN3 exhibits significantly higher binding affinity to cellulose surfaces compared to GFP alone, confirming FN3’s role as a cellulose-binding module. Control experiments ruled out nonspecific interactions, as GFP showed minimal adsorption. Second, isothermal titration calorimetry demonstrated strong lignin-BGL3 binding, correlating with a reduction in the thermophilic β-glucosidase’s specific activity, highlighting lignin’s inhibitory impact on catalytic efficiency. Third, molecular dynamics simulations of FN3 in lignin-containing environments showed no significant structural perturbations. However, sustained RMSD fluctuations indicated inherent conformational flexibility, attributed to dynamic loop. These findings establish FN3’s robust cellulose-binding capability, elucidate lignin’s dual role as a BGL3 inhibitor, and underscore FN3’s structural resilience in lignocellulosic matrices, advancing insights into enzyme engineering for biomass conversion.

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