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Holocellulase production by Aspergillus niger using raw sugarcane bagasse (rSCB) as the enzyme-inducing substrate is hampered by the intrinsic recalcitrance of this material. Here we report that mild hydrothermal pretreatment of rSCB increases holocellulase secretion by A. niger. Quantitative proteomic analysis revealed that pretreated solids (PS) induced a pronounced up-regulation of endoglucanases and cellobiohydrolases compared to rSCB, which resulted in a 10.1-fold increase in glucose release during SCB saccharification. The combined use of PS and pretreatment liquor (PL), referred to as whole pretreated slurry (WPS), as carbon source induced a more balanced up-regulation of cellulases, hemicellulases and pectinases and resulted in the highest increase (4.8-fold) in the release of total reducing sugars from SCB. The use of PL as the sole carbon source induced the modulation of A. niger’s secretome towards hemicellulose degradation. Mild pretreatment allowed the use of PL in downstream biological operations without the need for undesirable detoxification steps.

Lignin is an abundant cell wall component, and it has been used mainly for generating steam and electricity. Nevertheless, lignin valorization, i.e. the conversion of lignin into high value-added fuels, chemicals, or materials, is crucial for the full implementation of cost-effective lignocellulosic biorefineries. From this perspective, rapid screening methods are crucial for time-and resource-efficient development of novel microbial strains and enzymes with applications in the lignin biorefinery. The present review gives an overview of recent developments and applications of a vast arsenal of activity and sequence-based methodologies for uncovering novel microbial strains with ligninolytic potential, novel enzymes for lignin depolymerization and for unraveling the main metabolic routes during growth on lignin. Finally, perspectives on the use of each of the presented methods and their respective advantages and disadvantages are discussed.

An endo-β-1,4-xylanase (X22) was purified from crude extract of Emericella nidulans when cultivated on submerged fermentation using sugarcane bagasse as the carbon source. The purified protein was identified by mass spectrometry and was most active at pH and temperature intervals of 5.0-6.5 and 50-60°C, respectively. The enzyme showed half-lives of 40, 10 and 7 min at 28, 50 and 55°C, respectively, and pH 5.0. Apparent Km and Vmax values on soluble oat spelt xylan were 3.39 mg/mL and 230.8 IU/mg, respectively, while Kcat and Kcat/Km were 84.6 s(-1) and 25.0 s(-1) mg(-1) mL. Incubation with phenolic compounds showed that tannic acid and cinnamic acid had an inhibitory effect on X22 but no time-dependent deactivation. On the other hand, ferulic acid, 4-hydroxybenzoic acid, vanillin and p-coumaric acid did not show any inhibitory effect on X22 activity, although they changed X22 apparent kinetic parameters. Ethanol remarkably increased enzyme thermostability and apparent Vmax and Kcat values, even though the affinity and catalytic efficiency for xylan were lowered.

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Two new mass spectrometry methods, MALDI-TOF MS and hydrophilic interaction UHPLC-ESI-MS, were developed for the characterization of cellulose-active lytic polysaccharide monooxygenases, expanding the analytical toolbox for the study of these enzymes.

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