Study of non-catalytic domains in GH3-β-glucosidases in thermophilic bacterium Rhodothermus marinus- DSM 4252T and developing Engineered enzymes for biomass biodegradation

Ghosh, Sayani (2026) Study of non-catalytic domains in GH3-β-glucosidases in thermophilic bacterium Rhodothermus marinus- DSM 4252T and developing Engineered enzymes for biomass biodegradation. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Sayani Ghosh (19RS098)) 19RS098.pdf - Submitted Version Restricted to Repository staff only Download (4MB)

Abstract

Biofuel production from lignocellulosic biomass has emerged as a sustainable source of clean energy. Biomass is waste generated by plants or animals, and are utilised in the production of bioethanol, biobutanol, biomethane, etc. Lignocellulosic biomass is the largest source of cellulose that remains unperturbed and wasted. Cellulose is bound together with hemicellulose and lignin, joined by non-covalent hydrogen bonds, causing recalcitrance. The process of 2G bioethanol generation deconstructs this biomass by harsh pretreatment methods in biorefineries and loosens up the cellulose; dissolves and extracts the hemicellulose and lignin. The cellulose is broken down synergistically by the action of Endoglucanases, Cellobiohydrolases and β-glucosidases; also, hemicellulases (xylanases and mannanses) and lignin degrading enzymes (carbohydrate esterases and LPMOs). To increase the process efficiency for biomass deconstruction an optimized cocktail of selected enzymes or a production platform of cellulolytic host is required. This makes bioprospecting for better enzymes an urgent requirement. In this study we have heterologously cloned and characterized two GH3 β-glucosidases- RmGH3A and RmGH3B from thermophilic marine bacterium Rhodothermus marinus, to understand their potential in biomass degradation. We determined the optimum conditions for these enzymes, along with substrate profiling. Both these enzymes had very low activity on their natural substrate cellobiose, and had higher affinity towards β-1,2 and β-1,3 linked substrates. Two residues M179 and F270 were mutated by alanine scanning to check their role in substrate binding. The modular organisation of RmGH3A was explored. The PA14 domain was found to be attached to the active site tunnel and contained binding sites +1,+2 and +3 for longer chain oligosaccharide binding. RmGH3B without a PA14 domain was used to compare. We also investigated the lignin inhibition in β-glucosidases. Lignin heteropolymer was dissolved in water and tested for binding interaction with protein (in ITC), structural disorientation (by CD) and competitive binding of SfGFP-FN3 domain to Avicel was checked. This study highlights the modular architecture of GH3 β-glucosidases and explores the protein engineering strategies required for the design of better and more efficient enzymes.

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