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

Abstract

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.

Keywords

Enzymatic hydrolysis / Xylose-stimulated / Tolerant to biomass products / Homologous expression

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Funding

Fundação de Amparo à Pesquisa do Estado de São Paulo(2018/07522-6)

Fundação para a Ciência e a Tecnologia(POCI-01-0145-FEDER-032206)

Conselho Nacional de Desenvolvimento Científico e Tecnológico(310340/2021-7)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior(Financial Code 001)