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Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes found in viruses, archaea, and bacteria as well as eukaryotes, such as fungi, algae and insects, actively contributing to the degradation of different polysaccharides. In Aspergillus nidulans, LPMOs from family AA9 (AnLPMO9s), along with an AA3 cellobiose dehydrogenase (AnCDH1), are cosecreted upon growth on crystalline cellulose and lignocellulosic substrates, indicating their role in the degradation of plant cell wall components. Functional analysis revealed that three target LPMO9s (AnLPMO9C, AnLPMO9F and AnLPMO9G) correspond to cellulose-active enzymes with distinct regioselectivity and activity on cellulose with different proportions of crystalline and amorphous regions. AnLPMO9s deletion and overexpression studies corroborate functional data. The abundantly secreted AnLPMO9F is a major component of the extracellular cellulolytic system, while AnLPMO9G was less abundant and constantly secreted, and acts preferentially on crystalline regions of cellulose, uniquely displaying activity on highly crystalline algae cellulose. Single or double deletion of AnLPMO9s resulted in about 25% reduction in fungal growth on sugarcane straw but not on Avicel, demonstrating the contribution of LPMO9s for the saprophytic fungal lifestyle relies on the degradation of complex lignocellulosic substrates. Although the deletion of AnCDH1 slightly reduced the cellulolytic activity, it did not affect fungal growth indicating the existence of alternative electron donors to LPMOs. Additionally, double or triple knockouts of these enzymes had no accumulative deleterious effect on the cellulolytic activity nor on fungal growth, regardless of the deleted gene. Overexpression of AnLPMO9s in a cellulose-induced secretome background confirmed the importance and applicability of AnLPMO9G to improve lignocellulose saccharification. IMPORTANCE Fungal lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that boost plant biomass degradation in combination with glycoside hydrolases. Secretion of LPMO9s arsenal by Aspergillus nidulans is influenced by the substrate and time of induction. These findings along with the biochemical characterization of novel fungal LPMO9s have implications on our understanding of their concerted action, allowing rational engineering of fungal strains for biotechnological applications such as plant biomass degradation. Additionally, the role of oxidative players in fungal growth on plant biomass was evaluated by deletion and overexpression experiments using a model fungal system.

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BACKGROUND: Wood-decay basidiomycetes are effective for the degradation of highly lignified and recalcitrant plant substrates. The degradation of lignocellulosic materials by brown-rot strains is carried out by carbohydrate-active enzymes and non-enzymatic Fenton mechanism. Differences in the lignocellulose catabolism among closely related brown rots are not completely understood. Here, a multi-omics approach provided a global understanding of the strategies employed by L. sulphureus ATCC 52600 for lignocellulose degradation. RESULTS: The genome of Laetiporus sulphureus ATCC 52600 was sequenced and phylogenomic analysis supported monophyletic clades for the Order Polyporales and classification of this species within the family Laetiporaceae. Additionally, the plasticity of its metabolism was revealed in growth analysis on mono- and disaccharides, and polysaccharides such as cellulose, hemicelluloses, and polygalacturonic acid. The response of this fungus to the presence of lignocellulosic substrates was analyzed by transcriptomics and proteomics and evidenced the occurrence of an integrated oxidative-hydrolytic metabolism. The transcriptomic profile in response to a short cultivation period on sugarcane bagasse revealed 125 upregulated transcripts, which included CAZymes (redox enzymes and hemicellulases) as well as non-CAZy redox enzymes and genes related to the synthesis of low-molecular-weight compounds. The exoproteome produced in response to extended cultivation time on Avicel, and steam-exploded sugarcane bagasse, sugarcane straw, and Eucalyptus revealed 112 proteins. Contrasting with the mainly oxidative profile observed in the transcriptome, the secretomes showed a diverse hydrolytic repertoire including constitutive cellulases and hemicellulases, in addition to 19 upregulated CAZymes. The secretome induced for 7 days on sugarcane bagasse, representative of the late response, was applied in the saccharification of hydrothermally pretreated grass (sugarcane straw) and softwood (pine) by supplementing a commercial cocktail. CONCLUSION: This study shows the singularity of L. sulphureus ATCC 52600 compared to other Polyporales brown rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omics analysis reinforces the oxidative-hydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step.

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The aims of this work were to establish improved conditions for lipase production by Candida viswanathii using agroindustrial wastes in solid-state cultivation and to purify and evaluate the application of this enzyme for poultry fat hydrolysis. Mixed wheat bran plus spent barley grain (1 : 1, w/w) supplemented with 25.0% (w/w) olive oil increased the lipase production to 322.4%, compared to the initial conditions. When olive oil was replaced by poultry fat, the highest lipase production found at 40% (w/w) was 31.43 U/gds. By selecting, yeast extract supplementation (3.5%, w/w), cultivation temperature (30°C), and substrate moisture (40%, w/v), lipase production reached 157.33 U/gds. Lipase was purified by hydrophobic interaction chromatography, presenting a molecular weight of 18.5 kDa as determined by SDS-PAGE. The crude and purified enzyme showed optimum activity at pH 5.0 and 50°C and at pH 5.5 and 45°C, respectively. The estimated half-life at 50°C was of 23.5 h for crude lipase and 6.7 h at 40°C for purified lipase. Lipase presented high activity and stability in many organic solvents. Poultry fat hydrolysis was maximum at pH 4.0, reaching initial hydrolysis rate of 33.17 mmol/L/min. Thus, C. viswanathii lipase can be successfully produced by an economic and sustainable process and advantageously applied for poultry fat hydrolysis without an additional acidification step to recover the released fatty acids.

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In recent decades, increasing interest has been devoted to xylanolytic enzymes due to their potential use in many industrial processes. This study describes the production of xylanase, -xylosidase and -Larabinofuranosidase, belonging to the xylanolytic complex, by Penicillium janczewskii using brewer's spent grain as substrate for solid-state fermentation. The optimized conditions for high levels of xylanase, -xylosidase and -Larabinofuranosidase production were: 50% initial moisture, which was provided by Vogel's salt solution, seven days of cultivation at 20-30 °C. Fermentation enriched the bioproduct with some amino acids and did not add mycotoxins to it. The use of brewer's spent grain as substrate for fungal cultivation and enzyme production can both add value to this waste and reduce the production cost of xylanolytic enzymes.

Nas últimas décadas, há interesse crescente nas enzimas xilanolíticas devido à sua potencial utilização em muitos processos industriais. Este estudo descreve a produção de xilanase, -xilosidase e -Larabinofuranosidase, três enzimas do complexo xilanolítico, por Penicillium janczewski utilizando bagaço de cevada como substrato para fermentação em estado sólido. As condições selecionadas para a produção de elevados níveis de xilanase, - xilosidase e -L-arabinofuranosidase por esta linhagem fúngica foram 50% de umidade inicial, sendo esta fornecida por uma solução de sais de Vogel e cultivo por sete dias a 20-30 °C. O bioproduto fermentado foi enriquecido com alguns aminoácidos e se apresentou livre de micotoxinas. O uso do bagaço de cerveja como substrato para o cultivo de fungos e produção de enzimas não só pode agregar valor a esses resíduos, mas também reduzir o custo de produção de enzimas xilanolíticas.

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Background Two xylanases, Xyl I and Xyl II, were purified from the crude extracellular extract of a Trichoderma inhamatum strain cultivated in liquid medium with oat spelts xylan. Results The molecular masses of the purified enzymes estimated by SDS-PAGE and gel filtration were, respectively, 19 and 14 kDa for Xyl I and 21 and 14.6 kDa for Xyl II. The enzymes are glycoproteins with optimum activity at 50°C in pH 5.0-5.5 for Xyl I and 5.5 for Xyl II. The xylanases were very stable at 40°C and in the pH ranges from 4.5-6.5 for Xyl I and 4.0-8.0 for Xyl II. The ion Hg2+ and the detergent SDS strongly reduced the activity while 1,4-dithiothreitol stimulated both enzymes. The xylanases showed specificity for xylan, Km and Vmax of 14.5, 1.6 mg·mL-1 and 2680.2 and 462.2 U·mg of protein-1 (Xyl I) and 10.7, 4.0 mg·mL-1 and 4553.7 and 1972.7 U·mg of protein-1 (Xyl II) on oat spelts and birchwood xylan, respectively. The hydrolysis of oat spelts xylan released xylobiose, xylotriose, xylotetrose and larger xylooligosaccharides. Conclusions The enzymes present potential for application in industrial processes that require activity in acid conditions, wide-ranging pH stability, such as for animal feed, or juice and wine industries.

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In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn(2+) and the reducing agents ß -mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg(2+), Zn(2+), and Cu(2+) as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost.

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The production of extracellular xylanase, beta-xylosidase and alpha-l-arabinofuranosidase by the mesophilic fungus Penicillium janczewskii under submerged cultivation was investigated with different carbon sources. Optimization steps included studies of carbon source concentration, temperature of cultivation and initial pH of culture medium. The production of these enzymes was increased two times when cultures were supplemented with brewer's spent grain at 2% concentration, pH 6.0 and carried out at 25 degrees C. Under these optimized conditions were obtained xylanase activity of 15.19UmL(-1) and 23.54Umgprot(-1), beta-xylosidase activity of 0.16UmL(-1) and 0.25Umgprot(-1) and alpha-l-arabinofuranosidase activity of 0.67UmL(-1) and 1.04Umgprot(-1). Brewer's spent grain is a promising substrate for P. janczewskii growth and xylanolytic enzyme production, since it is the main by-product from the brewing industry, available in large amounts and at low-cost in many countries.

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