Recombinant GH3 β-glucosidase stimulated by xylose and tolerant to furfural and 5-hydroxymethylfurfural obtained from Aspergillus nidulans.

The β-glucosidase gene from Aspergillus nidulans FGSC A4 was cloned and overexpressed in the A. nidulans A773. The resulting purified β-glucosidase, named AnGH3, is a monomeric enzyme with a molecular weight of approximately 80 kDa, as confirmed by SDS-PAGE. Circular dichroism further validated its unique canonical barrel fold (β/α), a feature also observed in the 3D homology model of AnGH3. The most striking aspect of this recombinant enzyme is its robustness, as it retained 100% activity after 24 h of incubation at 45 and 50 ºC and pH 6.0. Even at 55 °C, it maintained 72% of its enzymatic activity after 6 h of incubation at the same pH. The kinetic parameters V max , K M , and Kcat/K M for ρ-nitrophenyl-β-D-glucopyranoside (ρNPG) and cellobiose were also determined. Using ρNPG, the enzyme demonstrated a V max of 212 U mg  - 1 , K M of 0.0607 mmol L  - 1 , and K cat /K M of 4521 mmol L  - 1  s  - 1 when incubated at pH 6.0 and 65 °C. The K M , V max , and K cat /K M using cellobiose were 2.7 mmol L  - 1 , 57 U mg  - 1 , and 27 mmol  -1  s  - 1 , respectively. AnGH3 activity was significantly enhanced by xylose and ethanol at concentrations up to 1.5 mol L  - 1 and 25%, respectively. Even in challenging conditions, at 65 °C and pH 6.0, the enzyme maintained its activity, retaining 100% and 70% of its initial activity in the presence of 200 mmol L  - 1 furfural and 5-hydroxymethylfurfural (HMF), respectively. The potential of this enzyme was further demonstrated by its application in the saccharification of the forage grass Panicum maximum, where it led to a 48% increase in glucose release after 24 h. These unique characteristics, including high catalytic performance, good thermal stability in hydrolysis temperature, and tolerance to elevated concentrations of ethanol, D-xylose, furfural, and HMF, position this recombinant enzyme as a promising tool in the hydrolysis of lignocellulosic biomass as part of an efficient multi-enzyme cocktail, thereby opening new avenues in the field of biotechnology and enzymology.