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

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 ß-glycosidase AfBgl1.3 from A. fumigatus after expression and purification in Pichia pastoris X-33. Structural analysis by circular dichroism revealed that increasing temperature destructured the enzyme; the apparent Tm value was 48.5 °C. The percentages of α-helix (36.3%) and ß-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 (IC50 = 2042 mM). The enzyme was active toward the substrates salicin (495.0 ± 49.0 U mg-1), pNPG (340.5 ± 18.6 U mg-1), cellobiose (89.3 ± 5.1 U mg-1), and lactose (45.1 ± 0.5 U mg-1), so it had broad specificity. The Vmax values were 656.0 ± 17.5, 706.5 ± 23.8, and 132.6 ± 7.1 U mg-1 toward p-nitrophenyl-ß-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-1) by about 26% after 12 h. Moreover, AfBgl1.3 acted synergistically with other Aspergillus fumigatus 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.

LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Boost Enzymatic Saccharification Activity of Cellulase Cocktail.

Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Since breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from Aspergillus fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (kcat of 147.9 s-1 and 0.64 s-1, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. AfCel6A and AfAA9_B association inhibited AfCel6A activity, an outcome that needs to be further investigated. However, AfCel6A or AfAA9_B addition boosted the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase Af-EGL7. Enzymatic cocktail supplementation with AfCel6A or AfAA9_B boosted the yield of fermentable sugars from complex substrates, especially sugarcane exploded bagasse, by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass.

Lytic Polysaccharide Monooxygenase from Aspergillus fumigatus can Improve Enzymatic Cocktail Activity During Sugarcane Bagasse Hydrolysis.

BACKGROUND: Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory enzymes that act synergistically with cellulases and which are increasingly being used in secondgeneration bioethanol production from biomasses. Several LPMOs have been identified in various filamentous fungi, including Aspergillus fumigatus. However, many LPMOs have not been characterized yet. OBJECTIVE: To report the role of uncharacterized A. fumigatus AfAA9_B LPMO. METHODS: qRT-PCR analysis was employed to analyze the LPMO gene expression profile in different carbon sources. The gene encoding an AfAA9_B (Afu4g07850) was cloned into the vector pET- 28a(+), expressed in the E. coli strain RosettaTM (DE3) pLysS, and purified by a Ni2+-nitrilotriacetic (Ni-NTA) agarose resin. To evaluate the specific LPMO activity, the purified protein peroxidase activity was assessed. The auxiliary LPMO activity was investigated by the synergistic activity in Celluclast 1.5L enzymatic cocktail. RESULTS: LPMO was highly induced in complex biomass like sugarcane bagasse (SEB), Avicel® PH-101, and CM-cellulose. The LPMO gene encoded a protein comprising 250 amino acids, without a CBM domain. After protein purification, the AfAA9_B molecular mass estimated by SDSPAGE was 35 kDa. The purified protein specific peroxidase activity was 8.33 ± 1.9 U g-1. Upon addition to Celluclast 1.5L, Avicel® PH-101 and SEB hydrolysis increased by 18% and 22%, respectively. CONCLUSION: A. fumigatus LPMO is a promising candidate to enhance the currently available enzymatic cocktail and can therefore be used in second-generation ethanol production.

Functional characterization of GH7 endo-1,4-ß-glucanase from Aspergillus fumigatus and its potential industrial application.

A gene encoding an endo-1,4-ß-glucanase (Afu6g01800) from A. fumigatus was cloned into the vector pET-28a(+) and expressed in the E. coli strain RosettaTM (DE3) pLysS. Sequence analysis indicated that the enzyme Af-EGL7 belonged to the GH7 family. The gene Af-egl7 encoded a protein comprising 460 amino acids, with a CBM1 domain at residues 424-460 and molecular mass of 52 kDa, as estimated by SDS-PAGE. This enzyme was optimally active at pH and temperatures ranging from 4.5 to 5.5 and from 40 to 60 °C, respectively. Mn2+ addition significantly enhanced the Af-EGL7 cellulase activity by 233%, whereas SDS addition fully inhibited this activity. Higher activity was observed toward ß-glucan than toward xyloglucan and CM-Cellulose, suggesting that the enzyme corresponds to a ß-1,3-1,4-glucanase. qRT-PCR in different culture media helped to establish the time-course expression profile. Different polysaccharides induced the gene Af-egl7 in a time-dependent manner; in the particular case of the substrate sugarcane exploded bagasse (SEB), Af-egl7 was induced 2500-fold. Upon addition to a commercial cellulase cocktail, Af-EGL7 significantly improved SEB saccharification, which suggested that the enzyme Af-EGL7 had great potential to hydrolyze complex biomass. From a biotechnological point of view, A. fumigatus Af-EGL7 is a promising candidate to enhance enzyme cocktails used in biorefineries such as consolidated bioprocessing.

Transcriptome and secretome analysis of Aspergillus fumigatus in the presence of sugarcane bagasse.

BACKGROUND: Sugarcane bagasse has been proposed as a lignocellulosic residue for second-generation ethanol (2G) produced by breaking down biomass into fermentable sugars. The enzymatic cocktails for biomass degradation are mostly produced by fungi, but low cost and high efficiency can consolidate 2G technologies. A. fumigatus plays an important role in plant biomass degradation capabilities and recycling. To gain more insight into the divergence in gene expression during steam-exploded bagasse (SEB) breakdown, this study profiled the transcriptome of A. fumigatus by RNA sequencing to compare transcriptional profiles of A. fumigatus grown on media containing SEB or fructose as the sole carbon source. Secretome analysis was also performed using SDS-PAGE and LC-MS/MS. RESULTS: The maximum activities of cellulases (0.032 U mL-1), endo-1,4-ß--xylanase (10.82 U mL-1) and endo-1,3-ß glucanases (0.77 U mL-1) showed that functional CAZymes (carbohydrate-active enzymes) were secreted in the SEB culture conditions. Correlations between transcriptome and secretome data identified several CAZymes in A. fumigatus. Particular attention was given to CAZymes related to lignocellulose degradation and sugar transporters. Genes encoding glycoside hydrolase classes commonly expressed during the breakdown of cellulose, such as GH-5, 6, 7, 43, 45, and hemicellulose, such as GH-2, 10, 11, 30, 43, were found to be highly expressed in SEB conditions. Lytic polysaccharide monooxygenases (LPMO) classified as auxiliary activity families AA9 (GH61), CE (1, 4, 8, 15, 16), PL (1, 3, 4, 20) and GT (1, 2, 4, 8, 20, 35, 48) were also differentially expressed in this condition. Similarly, the most important enzymes related to biomass degradation, including endoxylanases, xyloglucanases, ß-xylosidases, LPMOs, α-arabinofuranosidases, cellobiohydrolases, endoglucanases and ß-glucosidases, were also identified in the secretome. CONCLUSIONS: This is the first report of a transcriptome and secretome experiment of Aspergillus fumigatus in the degradation of pretreated sugarcane bagasse. The results suggest that this strain employs important strategies for this complex degradation process. It was possible to identify a set of genes and proteins that might be applied in several biotechnology fields. This knowledge can be exploited for the improvement of 2G ethanol production by the rational design of enzymatic cocktails.