From Tolerance to Thermostability: Exploring and Engineering Thermophilic Traits in Glycoside Hydrolases

Roy, Tannistha (2025) From Tolerance to Thermostability: Exploring and Engineering Thermophilic Traits in Glycoside Hydrolases. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Tannistha Roy (20MS089)) 20MS089_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (5MB)

Abstract

Thermophilic enzymes have been of immense interest in recent years as they have the potential to be used in the biofuel, textile, and food processing sectors. They are of interest because they can work best at elevated temperatures (more than 50 °C) while maintaining structural integrity and catalytic activity. During biofuel production, the breakdown of lignocellulosic biomass is a critical step, which is mainly catalyzed by a synergistic action of cellulolytic enzymes: endoglucanases, cellobiohydrolases (CBHs), and -glucosidases (BGLs). These enzymes need to be not only efficient but also thermophilic for industrial applications. In this research, we investigated the molecular mechanism of thermophilicity by characterizing a thermophilic GH6 CBH (G0S9B2) and improving the thermotolerance of a GH1 BGL from Agrobacterium tumefaciens (H0HC94). From bioinformatic analysis, we selected homologous sequences of the two enzymes and contrasted structural and sequence characteristics between thermophilic and mesophilic counterparts, though no obvious general pattern was observed. Product inhibition assays demonstrated that G0S9B2 had higher product tolerance among the GH6 CBHs. In H0HC94, we carried out structure-guided site-directed mutagenesis based on sequence alignment, B-factor analysis, and free energy changes predicted. Other rationally constructed mutations were also made. The generated mutants were tested by enzymatic assays to assess changes in optimum temperature, as well as in thermal stability using Differential Scanning Fluorimetry and Circular Dichroism. Two mutants increased optimum temperature but maintained their melting temperatures in effect, indicating a complex interplay between thermostability and thermotolerance. These results also raise further questions as to the determinants of protein thermophilicity. Future work will include molecular dynamics simulations, hydrogen bond network analysis, and other in-silico methods to further elucidate. Combinatorial mutations can also be pursued to improve both activity and stability for industrial application. iv

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