Recombinant GH3 β-glucosidase stimulated by xylose and tolerant to furfural and 5-hydroxymethylfurfural obtained from Aspergillus nidulans Diandra de Andrades, Robson C. Alnoch, Gabriela S. Alves, Jose C. S. Salgado, Paula Z. Almeida, et al. Bioresources and Bioprocessing, 2024 The β-glucosidase gene from Aspergillus nidulans FGSC A4 was cloned and overexpressed in the A. nidulans A773. The resulting purified β-glucosidase, named AnGH3, is a monomeric enzyme with a molecular weight of approximately 80 kDa, as confirmed by SDS-PAGE. Circular dichroism further validated its unique canonical barrel fold (β/α), a feature also observed in the 3D homology model of AnGH3. The most striking aspect of this recombinant enzyme is its robustness, as it retained 100% activity after 24 h of incubation at 45 and 50 ºC and pH 6.0. Even at 55 °C, it maintained 72% of its enzymatic activity after 6 h of incubation at the same pH. The kinetic parameters Vmax, KM, and Kcat/KM for ρ-nitrophenyl-β-D-glucopyranoside (ρNPG) and cellobiose were also determined. Using ρNPG, the enzyme demonstrated a Vmax of 212 U mg − 1, KM of 0.0607 mmol L − 1, and Kcat/KM of 4521 mmol L − 1 s − 1 when incubated at pH 6.0 and 65 °C. The KM, Vmax, and Kcat/KM using cellobiose were 2.7 mmol L − 1, 57 U mg − 1, and 27 mmol –1 s − 1, respectively. AnGH3 activity was significantly enhanced by xylose and ethanol at concentrations up to 1.5 mol L − 1 and 25%, respectively. Even in challenging conditions, at 65 °C and pH 6.0, the enzyme maintained its activity, retaining 100% and 70% of its initial activity in the presence of 200 mmol L − 1 furfural and 5-hydroxymethylfurfural (HMF), respectively. The potential of this enzyme was further demonstrated by its application in the saccharification of the forage grass Panicum maximum, where it led to a 48% increase in glucose release after 24 h. These unique characteristics, including high catalytic performance, good thermal stability in hydrolysis temperature, and tolerance to elevated concentrations of ethanol, D-xylose, furfural, and HMF, position this recombinant enzyme as a promising tool in the hydrolysis of lignocellulosic biomass as part of an efficient multi-enzyme cocktail, thereby opening new avenues in the field of biotechnology and enzymology.
Enhancing Laccase Production by Trametes hirsuta GMA-01 Using Response Surface Methodology and Orange Waste: A Novel Breakthrough in Sugarcane Bagasse Saccharification and Synthetic Dye Decolorization Guilherme Guimarães Ortolan, Alex Graça Contato, Guilherme Mauro Aranha, Jose Carlos Santos Salgado, Robson Carlos Alnoch, et al. Reactions, 2024 Trametes hirsuta GMA-01 was cultivated in a culture medium supplemented with orange waste, starch, wheat bran, yeast extract, and salts. The fungus produced several holoenzymes, but the laccase levels were surprisingly high. Given the highlighted applicability of laccases in various biotechnological areas with minimal environmental impact, we provided a strategy to increase its production using response surface methodology. The immobilization of laccase into ionic supports (CM-cellulose, DEAE-agarose, DEAE-cellulose, DEAE-Sephacel, MANAE-agarose, MANAE-cellulose, and PEI-agarose) was found to be efficient and recuperative, showcasing the technical prowess of research. The crude extract laccase (CE) and CM-cellulose-immobilized crude extract (ICE) showed optimum activity in acidic conditions (pH 3.0) and at 70 °C for the CE and 60 °C for the ICE. The ICE significantly increased thermostability at 60 °C for the crude extract, which retained 21.6% residual activity after 240 min. The CE and ICE were successfully applied to sugarcane bagasse hydrolysis, showing 13.83 ± 0.02 µmol mL−1 reducing sugars after 48 h. Furthermore, the CE was tested for dye decolorization, achieving 96.6%, 71.9%, and 70.8% decolorization for bromocresol green, bromophenol blue, and orcein, respectively (0.05% (w/v) concentration). The properties and versatility of T. hirsuta GMA-01 laccase in different biotechnological purposes are interesting and notable, opening several potential applications and providing valuable insights into the future of biotechnological development.
Microenzymes: Is There Anybody Out There? Jose Carlos Santos Salgado, Robson Carlos Alnoch, Maria de Lourdes Teixeira de Moraes Polizeli, Richard John Ward Protein Journal, 2024
Enhancing Xylanase Production from Aspergillus tamarii Kita and Its Application in the Bioconversion of Agro-Industrial Residues into Fermentable Sugars Using Factorial Design Jose Carlos Santos Salgado, Paulo Ricardo Heinen, Josana Maria Messias, Lummy Maria Oliveira-Monteiro, Mariana Cereia, et al. Fermentation, 2024 The endo-1,4-β-xylanases (EC 3.2.1.8) are the largest group of hydrolytic enzymes that degrade xylan, the major component of hemicelluloses, by catalyzing the hydrolysis of glycosidic bonds β-1,4 in this polymer, releasing xylooligosaccharides of different sizes. Xylanases have considerable potential in producing bread, animal feed, food, beverages, xylitol, and bioethanol. The fungus Aspergillus tamarii Kita produced xylanases in Adams’ media supplemented with barley bagasse (brewer’s spent grains), a by-product from brewery industries. The culture extract exhibited two xylanase activities in the zymogram, identified by mass spectrometry as glycosyl hydrolase (GH) families 10 and 11 (GH 10 and GH 11). The central composite design (CCD) showed excellent predictive capacity for xylanase production (23.083 U mL−1). Additionally, other enzyme activities took place during the submerged fermentation. Moreover, enzymatic saccharification based on a mixture design (MD) of three different lignocellulosic residues was helpful in the production of fermentable sugars by the A. tamarii Kita crude extract.
Biochemical Characterization of an Endoglucanase GH7 from Thermophile Thermothielavioides terrestris Expressed on Aspergillus nidulans Robson C. Alnoch, Jose C. S. Salgado, Gabriela S. Alves, Diandra de Andrades, Luana P. Meleiro, et al. Catalysts, 2023 Endoglucanases (EC 3.2.1.4) are important enzymes involved in the hydrolysis of cellulose, acting randomly in the β-1,4-glycosidic bonds present in the amorphous regions of the polysaccharide chain. These biocatalysts have been classified into 14 glycosyl hydrolase (GH) families. The GH7 family is of particular interest since it may act on a broad range of substrates, including cellulose, β-glucan, and xylan, an attractive feature for biotechnological applications, especially in the renewable energy field. In the current work, a gene from the thermophilic fungus Thermothielavioides terrestris, encoding an endoglucanase GH7 (TtCel7B), was cloned in the secretion vector pEXPYR and transformed into the high-protein-producing strain Aspergillus nidulans A773. Purified TtCel7B has a molecular weight of approximately 66 kDa, evidenced by SDS-PAGE. Circular dichroism confirmed the high β-strand content consistent with the canonical GH7 family β-jellyroll fold, also observed in the 3D homology model of TtCel7B. Biochemical characterization assays showed that TtCel7B was active over a wide range of pH values (3.5–7.0) and temperatures (45–70 °C), with the highest activity at pH 4.0 and 65 °C. TtCel7B also was stable over a wide range of pH values (3.5–9.0), maintaining more than 80% of its activity after 24 h. The KM and Vmax values in low-viscosity carboxymethylcellulose were 9.3 mg mL−1 and 2.5 × 104 U mg−1, respectively. The results obtained in this work provide a basis for the development of applications of recombinant TtCel7B in the renewable energy field.
Immobilization and Application of the Recombinant Xylanase GH10 of Malbranchea pulchella in the Production of Xylooligosaccharides from Hydrothermal Liquor of the Eucalyptus (Eucalyptus grandis) Wood Chips Robson C. Alnoch, Gabriela S. Alves, Jose C. S. Salgado, Diandra de Andrades, Emanuelle N. de Freitas, et al. International Journal of Molecular Sciences, 2022 Xylooligosaccharides (XOS) are widely used in the food industry as prebiotic components. XOS with high purity are required for practical prebiotic function and other biological benefits, such as antioxidant and inflammatory properties. In this work, we immobilized the recombinant endo-1,4-β-xylanase of Malbranchea pulchella (MpXyn10) in various chemical supports and evaluated its potential to produce xylooligosaccharides (XOS) from hydrothermal liquor of eucalyptus wood chips. Values >90% of immobilization yields were achieved from amino-activated supports for 120 min. The highest recovery values were found on Purolite (142%) and MANAE-MpXyn10 (137%) derivatives, which maintained more than 90% residual activity for 24 h at 70 °C, while the free-MpXyn10 maintained only 11%. In addition, active MpXyn10 derivatives were stable in the range of pH 4.0–6.0 and the presence of the furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan of various sources. Maximum values were observed for birchwood xylan at 8.6 mg mL−1 and wheat arabinoxylan at 8.9 mg mL−1, using Purolite-MpXyn10. Its derivative was also successfully applied in the hydrolysis of soluble xylan present in hydrothermal liquor, with 0.9 mg mL−1 of XOS after 3 h at 50 °C. This derivative maintained more than 80% XOS yield after six cycles of the assay. The results obtained provide a basis for the application of immobilized MpXyn10 to produce XOS with high purity and other high-value-added products in the lignocellulosic biorefinery field.
Immobilization of Thermophilic GH11 Xylanase (XylHb) of Humicola brevis var. thermoidea on MANAE-Agarose Enhances Catalytic Performance and Operational Stability for XOS Production AP Almeida, TVC Oliveira, RC Alnoch, JCS Salgado, D de Andrades, ... Biocatalysis and Agricultural Biotechnology, 104000 , 2026 2026
Assessing process variable effects on laccase-mediated treatment for enhanced saccharification efficiency M Michelin, DG Gomes, EN Freitas, RC Alnoch, JCS Salgado, E Coelho, ... Industrial Crops and Products 233, 121367 , 2025 2025
Homologous expression, purification, and characterization of a recombinant acetylxylan esterase from Aspergillus nidulans GS Alves, D de Andrades, JCS Salgado, CB Mariano, GL Berto, F Segato, ... International journal of biological macromolecules 280, 135816 , 2024 2024 Citations: 4
Enhancing Laccase Production by Trametes hirsuta GMA-01 Using Response Surface Methodology and Orange Waste: A Novel Breakthrough in Sugarcane … GG Ortolan, AG Contato, GM Aranha, JCS Salgado, RC Alnoch, ... Reactions 5 (3), 635-650 , 2024 2024 Citations: 6
Recombinant GH3 β-glucosidase stimulated by xylose and tolerant to furfural and 5-hydroxymethylfurfural obtained from Aspergillus nidulans D de Andrades, RC Alnoch, GS Alves, JCS Salgado, PZ Almeida, ... Bioresources and Bioprocessing 11 (1), 77 , 2024 2024 Citations: 8
Microenzymes: Is There Anybody Out There? JCS Salgado, RC Alnoch, MLTM Polizeli, RJ Ward The Protein Journal 43 (3), 393-404 , 2024 2024 Citations: 1
Enhancing Xylanase Production from Aspergillus tamarii Kita and Its Application in the Bioconversion of Agro-Industrial Residues into Fermentable Sugars Using … JCS Salgado, PR Heinen, JM Messias, LM Oliveira-Monteiro, M Cereia, ... Fermentation 10 (5), 241 , 2024 2024 Citations: 3
Synthetic carbohydrate-binding module-endogalacturonase chimeras increase catalytic efficiency and saccharification of lignocellulose residues S Carli, L Parras Meleiro, JCS Salgado, RJ Ward Biomass Conversion and Biorefinery 14 (5), 6369-6380 , 2024 2024 Citations: 10
A double square slot antenna for 5G small-area wireless access points in street furniture TES Oliveira, JR Reis, T Fernandes, S Madail, J Salgado, ... 2023 International Wireless Communications and Mobile Computing (IWCMC), 273-277 , 2023 2023 Citations: 4
A wideband reduced form factor antenna for 5G SAWAP applications TES Oliveira, JR Reis, TR Fernandes, S Madail, J Salgado, ... 2023 Joint European Conference on Networks and Communications & 6G Summit … , 2023 2023 Citations: 1
Biochemical Characterization of an Endoglucanase GH7 from Thermophile Thermothielavioides terrestris Expressed on Aspergillus nidulans RC Alnoch, JCS Salgado, GS Alves, D de Andrades, LP Meleiro, ... Catalysts 13 (3), 582 , 2023 2023 Citations: 11
Immobilization and Application of the Recombinant Xylanase GH10 of Malbranchea pulchella in the Production of Xylooligosaccharides from Hydrothermal Liquor of the … RC Alnoch, GS Alves, JCS Salgado, D de Andrades, EN Freitas, ... International Journal of Molecular Sciences 23 (21), 13329 , 2022 2022 Citations: 18
Biochemical characterization and biological properties of mycelium extracts from Lepista sordida GMA-05 and Trametes hirsuta GMA-01: new mushroom strains isolated in Brazil GM Aranha, AG Contato, JCS Salgado, TB de Oliveira, KM Retamiro, ... Brazilian Journal of Microbiology 53 (1), 349-358 , 2022 2022 Citations: 20
Covalent Immobilization of Chondrostereum purpureum Endopolygalacturonase on Ferromagnetic Nanoparticles: Catalytic Properties and Biotechnological … S Carli, JCS Salgado, LP Meleiro, RJ Ward Applied biochemistry and biotechnology 194 (2), 848-861 , 2022 2022 Citations: 5
Enhanced hydrolytic efficiency of an engineered CBM11-glucanase enzyme chimera against barley β-D-glucan extracts GP Furtado, S Carli, LP Meleiro, JCS Salgado, RJ Ward Food chemistry 365, 130460 , 2021 2021 Citations: 14
Challenges of biomass utilization for bioenergy in a climate change scenario EN Freitas, JCS Salgado, RC Alnoch, AG Contato, E Habermann, ... Biology 10 (12), 1277 , 2021 2021 Citations: 79
Polizeli, MdLTM Challenges of Biomass Utilization for Bioenergy in a Climate Change Scenario EN De Freitas, JCS Salgado, RC Alnoch, AG Contato, E Habermann, ... Biology 10, 1277 , 2021 2021 Citations: 4
Heterologous production and biochemical characterization of a new highly glucose tolerant GH1 β-glucosidase from Anoxybacillus thermarum PZ de Almeida, TB de Oliveira, RC de Lucas, JCS Salgado, MM Perez, ... Process Biochemistry 99, 1-8 , 2020 2020 Citations: 14
A Halotolerant Endo-1,4-β-Xylanase from Aspergillus clavatus with Potential Application for Agroindustrial Residues Saccharification TM Pasin, JCS Salgado, ASA Scarcella, TB de Oliveira, RC de Lucas, ... Applied biochemistry and biotechnology 191 (3), 1111-1126 , 2020 2020 Citations: 41
A novel Trichoderma reesei mutant RP698 with enhanced cellulase production JCR Silva, JCS Salgado, AC Vici, RJ Ward, MLTM Polizeli, ... Brazilian journal of microbiology 51 (2), 537-545 , 2020 2020 Citations: 36
Challenges of biomass utilization for bioenergy in a climate change scenario EN Freitas, JCS Salgado, RC Alnoch, AG Contato, E Habermann, ... Biology 10 (12), 1277 , 2021 2021 Citations: 79
A Halotolerant Endo-1,4-β-Xylanase from Aspergillus clavatus with Potential Application for Agroindustrial Residues Saccharification TM Pasin, JCS Salgado, ASA Scarcella, TB de Oliveira, RC de Lucas, ... Applied biochemistry and biotechnology 191 (3), 1111-1126 , 2020 2020 Citations: 41
A novel Trichoderma reesei mutant RP698 with enhanced cellulase production JCR Silva, JCS Salgado, AC Vici, RJ Ward, MLTM Polizeli, ... Brazilian journal of microbiology 51 (2), 537-545 , 2020 2020 Citations: 36
Engineering the GH1 β-glucosidase from Humicola insolens : Insights on the stimulation of activity by glucose and xylose LP Meleiro, JCS Salgado, RF Maldonado, S Carli, LAB Moraes, RJ Ward, ... PLoS One 12 (11), e0188254 , 2017 2017 Citations: 36
A Neurospora crassa ÿ-glucosidase with potential for lignocellulose hydrolysis shows strong glucose tolerance and stimulation by glucose and xylose LP Meleiro, JCS Salgado, RF Maldonado, JS Alponti, ALRL Zimbardi, ... Journal of Molecular Catalysis B: Enzymatic 122, 131-140 , 2015 2015 Citations: 29
Production of omegas-6 and 9 from the hydrolysis of açaí and buriti oils by lipase immobilized on a hydrophobic support MM Pérez, ECS Gonçalves, JCS Salgado, MS Rocha, PZ Almeida, ... Molecules 23 (11), 3015 , 2018 2018 Citations: 24
Potential biodiesel production from Brazilian plant oils and spent coffee grounds by Beauveria bassiana lipase 1 expressed in Aspergillus nidulans A773 using different … ECS Gonçalves, MM Perez, AC Vici, JCS Salgado, M de Souza Rocha, ... Journal of Cleaner Production 256, 120513 , 2020 2020 Citations: 21
Biochemical characterization and biological properties of mycelium extracts from Lepista sordida GMA-05 and Trametes hirsuta GMA-01: new mushroom strains isolated in Brazil GM Aranha, AG Contato, JCS Salgado, TB de Oliveira, KM Retamiro, ... Brazilian Journal of Microbiology 53 (1), 349-358 , 2022 2022 Citations: 20
Immobilization and Application of the Recombinant Xylanase GH10 of Malbranchea pulchella in the Production of Xylooligosaccharides from Hydrothermal Liquor of the … RC Alnoch, GS Alves, JCS Salgado, D de Andrades, EN Freitas, ... International Journal of Molecular Sciences 23 (21), 13329 , 2022 2022 Citations: 18
Fungal lipases: versatile tools for white biotechnology MM Pérez, ECS Gonçalves, AC Vici, JCS Salgado, ... Recent Advancement in White Biotechnology Through Fungi: Volume 1: Diversity … , 2019 2019 Citations: 16
Purification and biochemical characterization of glucose–cellobiose-tolerant cellulases from S cytalidium thermophilum JCR Silva, LHS Guimarães, JCS Salgado, RPM Furriel, MLTM Polizeli, ... Folia microbiologica 58 (6), 561-568 , 2013 2013 Citations: 16
Enhanced hydrolytic efficiency of an engineered CBM11-glucanase enzyme chimera against barley β-D-glucan extracts GP Furtado, S Carli, LP Meleiro, JCS Salgado, RJ Ward Food chemistry 365, 130460 , 2021 2021 Citations: 14
Heterologous production and biochemical characterization of a new highly glucose tolerant GH1 β-glucosidase from Anoxybacillus thermarum PZ de Almeida, TB de Oliveira, RC de Lucas, JCS Salgado, MM Perez, ... Process Biochemistry 99, 1-8 , 2020 2020 Citations: 14
Biochemical Characterization of an Endoglucanase GH7 from Thermophile Thermothielavioides terrestris Expressed on Aspergillus nidulans RC Alnoch, JCS Salgado, GS Alves, D de Andrades, LP Meleiro, ... Catalysts 13 (3), 582 , 2023 2023 Citations: 11
Synthetic carbohydrate-binding module-endogalacturonase chimeras increase catalytic efficiency and saccharification of lignocellulose residues S Carli, L Parras Meleiro, JCS Salgado, RJ Ward Biomass Conversion and Biorefinery 14 (5), 6369-6380 , 2024 2024 Citations: 10
Recombinant GH3 β-glucosidase stimulated by xylose and tolerant to furfural and 5-hydroxymethylfurfural obtained from Aspergillus nidulans D de Andrades, RC Alnoch, GS Alves, JCS Salgado, PZ Almeida, ... Bioresources and Bioprocessing 11 (1), 77 , 2024 2024 Citations: 8
Enhancing Laccase Production by Trametes hirsuta GMA-01 Using Response Surface Methodology and Orange Waste: A Novel Breakthrough in Sugarcane … GG Ortolan, AG Contato, GM Aranha, JCS Salgado, RC Alnoch, ... Reactions 5 (3), 635-650 , 2024 2024 Citations: 6
Covalent Immobilization of Chondrostereum purpureum Endopolygalacturonase on Ferromagnetic Nanoparticles: Catalytic Properties and Biotechnological … S Carli, JCS Salgado, LP Meleiro, RJ Ward Applied biochemistry and biotechnology 194 (2), 848-861 , 2022 2022 Citations: 5
Antidiabetic and toxicological evaluations of naringenin in normoglycaemic and NIDDM rat models and its implicationson extra-pancreatic glucose regulation RR O-Andrade, JCS Salgado, GN Vázquez, SP Webster, M Binnie, ... Diabetes ObesMetab 10, 1097-1104 , 2008 2008 Citations: 5
