Identification of a thermostable GH6 family cellulase from chaetomium thermophilum exhibiting high cellobiose and ionic liquid tolerance

Efficient conversion of lignocellulosic biomass into fermentable sugars requires cellulases that are both thermostable and tolerant to inhibitors such as ionic liquids (ILs). Thermostable enzymes are particularly valuable for industrial applications, as they maintain activity at elevated temperatures for extended periods, improve product yield, and reduce process costs. In this study, we cloned and characterized CtCel6C, a GH6 family cellulase, from the thermophilic fungus Thermochaetoides thermophila (Chaetomium thermophilum). CtCel6C exhibited a specific activity of 28 U/mg on carboxymethyl cellulose (CMC-Na) at 55 °C and pH 6.0, retaining 90 % of its specific activity after 20 h at optimum pH and temperature (55 °C and pH 6.0). CtCel6C demonstrated broad substrate specificity, effectively hydrolyzing oligosaccharides, soluble substrates (CMC and barley β-glucan), and insoluble substrates such as Avicel, consistently producing cellobiose as the sole product and outperforming other GH6 cellobiohydrolases from C. thermophilum. Despite having an open active-site cleft due to a lack of a fifteen-residue stretch in the C-terminal loop, CtCel6C retains the biochemical characteristics of a processive cellobiohydrolase rather than those of an endoglucanase from the GH6 family. The enzyme also exhibited high tolerance to cellobiose, glucose, and to 20 % (v/v) of the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C₂mim][OAc]), in both McIlvaine buffer (pH 6.0) and seawater. The remarkable IL tolerance of CtCel6C makes it a promising candidate for integration into industrial enzyme cocktails for biomass saccharification.