No abstract

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.

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.