Lignocellulosic biomass can be readily obtained from plants, seaweeds, and agricultural waste and is a beneficial resource for producing biofuels and other valuable bio-products. The major component of lignocellulose biomass is cellulose (40–60 %), a polymer of repeated units of glucose (up to 15000) linked by a β-1,4-glycosidic bond [1]. The other significant constituents of lignocellulose are lignin and hemicellulose, which form a protective barrier around the cellulose in the cell wall and must be separated from cellulose for efficient enzymatic treatment. Since the high crystallinity of cellulose decreases the efficiency of enzymatic hydrolysis, pretreatment methods, such as by ionic liquid (IL), help to separate the lignin and hemicellulose from cellulose, reduce cellulose crystallinity, and enhance solubility and the efficiency of cellulose hydrolysis to sugar for further downstream applications such as the production of biofuels [2], [3]. Although ionic liquids are environmentally friendly, their application in biomass pretreatment and hydrolysis faces challenges. One key issue is the limited availability of IL-compatible cellulases. Addressing this may require discovering or genetically engineering cellulases that tolerate ionic liquids.

Enzymatic depolymerization of cellulose requires a minimum of three enzymes: endo-1,4-β-glucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21) [4]. Based on sequence similarities and structural characteristics, endoglucanases are classified into glycoside hydrolase (GH) families GH5, GH6, GH7, GH9, GH12, GH45, and GH74, while cellobiohydrolases (CBH) belong to GH6, GH7, and GH48 [5]. Notably, GH6 and GH7 families include members with either cellobiohydrolase or endoglucanase activity. Structurally, cellobiohydrolases feature a tunnel-shaped active site, where long, flexible loops form a roof over the tunnel [6]. As cellulose threads through this tunnel, the enzyme cleaves the glycosidic bond between the second and third glucose units from one end, releasing cellobiose. In contrast, endoglucanases have an open cleft active site, allowing them to bind randomly along the cellulose chain and produce oligosaccharides [7]. Cellobiohydrolase is particularly crucial in cellulose hydrolysis, as it processively degrades cellulose chains or the products generated by endoglucanases into cellobiose as the primary product, which is then converted to glucose by β-glucosidase. According to the CAZy database, fungal GH7 CBHs hydrolyze cellulose from the reducing end, whereas GH6 CBHs act on the non-reducing end, yielding cellobiose, cellotriose, and glucose as major products. Although GH7 cellulases have been well studied, GH6 enzymes remain less understood, particularly the mechanism of product formation, product inhibition, processivity, and IL tolerance.

Thermostable cellulases offer key advantages at high temperatures, including reduced contamination risk, lower substrate viscosity, and improved mixing efficiency [8]. Most endoglucanases from GH5, GH7, and GH12 are highly thermostable, while GH7 CBH and GH1 BG show high thermostability [9]. Most reported GH6 cellulases across mesophilic and thermophilic organisms exhibit optimum activity between 50 – 80 °C, and typically lose half of their initial activity within an hour (Table S1). This underscores the need to discover more robust and thermostable GH6 cellulases.

The thermophilic filamentous fungus Chaetomium thermophilum thrives at 45–55 °C and can withstand temperatures up to 60 °C [10]. It produces a variety of cellulases, including β-1,4-endoglucanase (CTendo45), GH7 cellobiohydrolase, and GH6 cellobiohydrolase [11], [12], [13], [14]. Although the C. thermophilum secretome contains relatively few cellulases, it efficiently degrades cellulose at high temperatures, likely due to the effectiveness of its encoded cellulases [15]. Previously, a C. thermophilum CBHI expression in Trichoderma reesei enhanced the latter's cellulose degradation efficiency [16]. Interestingly, two GH6 enzymes from C. thermophilum, CtCel6A and CtCel6, exhibit significantly different thermostability [13], [17]. While CtCel6A retains 70 % activity after 48 h at its Topt, CtCel6 loses 30 % of its activity within one hour at its Topt. This suggests that not all enzymes encoded in thermophilic organisms necessarily exhibit high thermostability. Engineering stable enzymes is challenging since the molecular basis of thermostability remains poorly understood. Thus, prospecting natural thermophiles and enzymes may be more effective than attempting laboratory-directed enhancement of thermostability.

Towards the search for thermostable cellulase, we characterized the third GH6 family cellulase, CtCel6C, from C. thermophilum and compared it to the previously characterized CtCel6A and CtCel6. CtCel6C efficiently hydrolyzes soluble and insoluble substrates at 55 °C for over 20 h, producing cellobiose as the sole product. Notably, cellobiose does not inhibit CtCel6C, and the enzyme tolerates high concentrations of ionic liquids, a common biomass pretreatment solvent. All of these properties make CtCel6C a strong candidate for biorefinery applications.