Fungal β-Glucosidases with dual Activity: Linking biomass saccharification to cellulase induction in Trichoderma...

The cellulolytic system of Trichoderma reesei depends on β-glucosidases as central enzymes completing biomass hydrolysis and generating inducers such as sophorose. Three β-glucosidases from Aspergillus clavatus (AC), Penicillium oxalicum (PO) and Talaromyces stipitatus (TS) were expressed in Komagataella phaffii and characterized for parameters relevant to biomass conversion and inducer formation. PO and TS showed higher thermostability between 60 and 70 °C, whereas AC was inactivated above 50 °C. All variants were glucose inhibited, with AC least affected (K_i = 8.4 mM), followed by PO (K_i = 6 mM) and TS (K_i = 2.3 mM). Supplementation of Celluclast® with TS increased glucose release from cellulose and wheat straw and reached levels comparable to Cellic®CTec2 on wheat straw after 24 h. All enzymes catalyzed condensation reactions and produced up to 55 g/L disaccharides, with TS yielding the highest sophorose titers at 700 g/L glucose. Induction performance was assessed using T.reesei RUT-C30 cultivated with glucose, a TS derived disaccharide solution or steam-ex wheat straw hydrolysate. For each enzyme class, the highest activity across all conditions was defined as 100 %. The disaccharide solution generated the strongest cellulase response, while glucose yielded only 26 % BGL and 20 % CBH1 activity relative to these maxima, and hydrolysates produced intermediate levels of 65 % BGL and 24 % CBH1 activity while concurrently generating the highest hemicellulolytic activities, set to 100 %. Proteomics confirmed that the disaccharide solution upregulated core cellulases (EGL1 +2.80, CBH1 +1.66, EGL5 +3.16), whereas hydrolysates enriched CBH2 (-2.02), GH11 xylanases (-3.27) and GH3 β-xylosidases (-6.45, -2.49).