Characterization of a New Glucose-Tolerant GH1 β-Glycosidase from Aspergillus fumigatus with Transglycosylation Activity

• Published in: International journal of molecular sciences 2023-02, Vol.24 (5), p.4489
• Main Authors: Pereira, Lucas Matheus Soares, Bernardi, Aline Vianna, Gerolamo, Luis Eduardo, Pedersoli, Wellington Ramos, Carraro, Cláudia Batista, Silva, Roberto do Nascimento, Uyemura, Sergio Akira, Dinamarco, Taísa Magnani
• Format: Article
• Language: English
• Subjects: Aspergillus fumigatus; Aspergillus fumigatus - metabolism; Bagasse; beta-Glucosidase - metabolism; Biofuels; Biomass; Cellobiose; Cellulose; Circular dichroism; Dichroism; Drug tolerance; enzymatic hydrolysis; Enzymes; Ethanol; Ethanol - metabolism; Glucose; Glucose - metabolism; glucose stimulation; Glucose tolerance; Glycosidases; Glycoside Hydrolases - metabolism; Hydrogen-Ion Concentration; Hydrolysis; Lactose; Lignocellulose; p-Nitrophenyl-b-D-glucopyranoside; Proteins; Saccharification; Salicin; Structural analysis; Sugar; Sugarcane; transglycosylation activity; β-glycosidase
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24054489

Concern over environmental impacts has spurred many efforts to replace fossil fuels with biofuels such as ethanol. However, for this to be possible, it is necessary to invest in other production technologies, such as second generation (2G) ethanol, in order to raise the levels of this product and meet the growing demand. Currently, this type of production is not yet economically feasible, due to the high costs of the enzyme cocktails used in saccharification stage of lignocellulosic biomass. In order to optimize these cocktails, the search for enzymes with superior activities has been the goal of several research groups. For this end, we have characterized the new β-glycosidase AfBgl1.3 from after expression and purification in X-33. Structural analysis by circular dichroism revealed that increasing temperature destructured the enzyme; the apparent T value was 48.5 °C. The percentages of α-helix (36.3%) and β-sheet (12.4%) secondary structures at 25 °C were predicted. Biochemical characterization suggested that the optimal conditions for AfBgl1.3 were pH 6.0 and temperature of 40 °C. At 30 and 40 °C, the enzyme was stable and retained about 90% and 50% of its activity, respectively, after pre-incubation for 24 h. In addition, the enzyme was highly stable at pH between 5 and 8, retaining over 65% of its activity after pre-incubation for 48 h. AfBgl1.3 co-stimulation with 50-250 mM glucose enhanced its specific activity by 1.4-fold and revealed its high tolerance to glucose (IC = 2042 mM). The enzyme was active toward the substrates salicin (495.0 ± 49.0 U mg ), pNPG (340.5 ± 18.6 U mg ), cellobiose (89.3 ± 5.1 U mg ), and lactose (45.1 ± 0.5 U mg ), so it had broad specificity. The V values were 656.0 ± 17.5, 706.5 ± 23.8, and 132.6 ± 7.1 U mg toward -nitrophenyl-β-D-glucopyranoside (pNPG), D-(-)-salicin, and cellobiose, respectively. AfBgl1.3 displayed transglycosylation activity, forming cellotriose from cellobiose. The addition of AfBgl1.3 as a supplement at 0.9 FPU/g of cocktail Celluclast 1.5L increased carboxymethyl cellulose (CMC) conversion to reducing sugars (g L ) by about 26% after 12 h. Moreover, AfBgl1.3 acted synergistically with other cellulases already characterized by our research group-CMC and sugarcane delignified bagasse were degraded, releasing more reducing sugars compared to the control. These results are important in the search for new cellulases and in the optimization of enzyme cocktails for saccharification.