Characterization and engineering of CBHII from Chaetomium thermophilum and its application in efficient degradation of lignocellulosic biomass

Pooja, . (2025) Characterization and engineering of CBHII from Chaetomium thermophilum and its application in efficient degradation of lignocellulosic biomass. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Lignocellulosic biomass, the most abundant renewable source of carbon, is considered a sustainable feedstock for biofuel production. Its major component cellulose requires synergistic action of cellulases, namely endo-1,4-β-glucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21), which must remain stable and active under industrially relevant conditions. Chaetomium thermophilum, a thermophilic fungus, has emerged as a promising system and its produces thermostable enzymes. Although this organism encodes a relatively small number of cellobiohydrolases (CBHs), they play a pivotal role in crystalline cellulose degradation. According to the CAZy database, CBHs are classified into GH7 (CBHI), which attack cellulose chains from the reducing end, and GH6 (CBHII), which act from the non-reducing end. While GH7 CBHs have been extensively investigated, GH6 CBHs remain comparatively underexplored, particularly regarding thermostability, product inhibition, and tolerance to harsh industrial solvents. In this study, we characterised CtCel6C, a previously uncharacterized GH6 CBH from C. thermophilum, heterologously expressed in Escherichia coli. CtCel6C displayed exceptional thermostability, broad substrate specificity, and high resistance to glucose and cellobiose. The enzyme released cellobiose as a major product, demonstrating strict processive hydrolysis despite lacking the 15-amino acid stretch from the C-terminal loop. Comparative analysis with another GH6 CBH, CtCel6, revealed that both enzymes exhibited tight binding to cellotriose, a potent inhibitor of GH6 CBHs. CtCel6C showed stronger affinity towards cellotriose, and a non-conserved tyrosine residue near the –2 subsite enhanced activity on crystalline cellulose and also increased the resistance for cellotriose. Cysteine mutagenesis experiments confirmed that conserved disulfide bonds are essential for stability, whereas non-conserved free cysteines in CtCel6 affect the activity and stability. Furthermore, both GH6 CBHs retained significant activity in ionic liquids such as [C₂mim][OAc] and in seawater, highlighting their robustness under pretreatment and saline conditions. Overall, this study highlights CtCel6C as a structurally distinct, thermostable, and inhibitor-tolerant CBHII with potential application in biomass conversion. These findings provide valuable insights into the functional diversity of GH6 enzymes in C. thermophilum and lay the groundwork for protein engineering strategies to enhance cellulase performance in industrial biorefineries.

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