LPMO <i>Af</i>AA9_B and Cellobiohydrolase <i>Af</i>Cel6A from <i>A. fumigatus</i> Boost Enzymatic Saccharification Activity of Cellulase Cocktail

• Main Authors: Aline Vianna Bernardi, Luis Eduardo Gerolamo, Paula Fagundes de Gouvêa, Deborah Kimie Yonamine, Lucas Matheus Soares Pereira, Arthur Henrique Cavalcante de Oliveira, Sérgio Akira Uyemura, Taisa Magnani Dinamarco
• Format: Article
• Language: English
• Published: MDPI AG 2021-12-01
• Series: International Journal of Molecular Sciences
• Subjects: GH6 cellobiohydrolase; AA9 LPMO; lignocellulose hydrolysis; bioethanol
• Online Access: https://www.mdpi.com/1422-0067/22/1/276
• ISSN: 1661-6596; 1422-0067

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 (<i>Af</i>Cel6A) and lytic polysaccharide monooxygenase LPMO (<i>Af</i>AA9_B) from <i>Aspergillus fumigatus</i> after they were expressed in <i>Pichia pastoris</i> and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (<i>k</i>_cat of 147.9 s⁻¹ and 0.64 s⁻¹, respectively). Surprisingly, when combined, <i>Af</i>Cel6A and <i>Af</i>AA9_B did not act synergistically. <i>Af</i>Cel6A and <i>Af</i>AA9_B association inhibited <i>Af</i>Cel6A activity, an outcome that needs to be further investigated. However, <i>Af</i>Cel6A or <i>Af</i>AA9_B addition boosted the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase <i>Af</i>-EGL7. Enzymatic cocktail supplementation with <i>Af</i>Cel6A or <i>Af</i>AA9_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 <i>Af</i>AA9_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 <i>Af</i>AA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass.