Characterization of two GH10 enzymes with ability to hydrolyze pretreated Sorghum bicolor bagasse

Applied Microbiology and Biotechnology (2025) 109:104 https://doi.org/10.1007/s00253-025-13484-4 RESEARCH Characterization of two GH10 enzymes with ability to hydrolyze pretreated Sorghum bicolor bagasse Camila Bruno Baron1,2 · María Laura Mon1 · Rubén Marrero Díaz de Villegas1 · Andrea Cattaneo3 · Paola Di Donato3,4 · Annarita Poli3 · Maria Emilia Negri5 · Mariana Alegre5,6 · Marcelo A. Soria7 · María Cecilia Rojo8,9 · Mariana Combina8,9 · Ilaria Finore3 · Paola M. Talia1,2 Received: 21 February 2025 / Revised: 3 April 2025 / Accepted: 4 April 2025 © The Author(s) 2025 Abstract In this study, we characterized two novel enzymes of the glycoside hydrolase family 10 (GH10), Xyl10 C and Xyl10E, identified in the termite gut microbiome. The activities of both enzymes were assayed using beechwood xylan, barley β-glucan, and pretreated Sorghum bicolor bagasse (SBB) as substrates. Both enzymes, assessed individually and in combination, showed activity on beechwood xylan and pretreated SBB, whereas Xyl10E also showed activity on barley β-glucan. The composition of pretreated SBB mainly consisted of xylose and arabinose content. Purified Xyl10 C showed optimum xylanase activity in the pH range 7.0–8.0 and at a temperature of 50–60 °C, while Xyl10E was active at a wider pH range (5.0–10.0) and at 50 °C. The residual activities of Xyl10 C and Xyl10E after 8 h of incubation at 40 °C were 85% and 70%, respectively. The enzymatic activity of Xyl10 C increased to 115% in the presence of 5 M NaCl, was only inhibited in the presence of 0.5% sodium dodecyl sulfate (SDS), and decreased with β-mercaptoethanol. The xylanase and glucanase activities of Xyl10E were inhibited only in the presence of MnSO4, NaCl, and SDS. The main hydrolysis enzymatic product of Xyl10 C and Xyl10E on pretreated SBB was xylobiose. In addition, the xylo-oligosaccharides produced by xylanase Xyl10E on pretreated SBB demonstrated promising antioxidant activity. Thus, the hydrolysis products using Xyl10E on pretreated SBB indicate potential for antioxidant activity and other valuable industrial applications. Key points • Two novel GH10 xylanases from the termite gut microbiome were characterized. • Xylo-oligosaccharides obtained from sorghum bagasse exhibited antioxidant potential. • Both enzymes and their hydrolysis product have potential to add value to agro-waste. Keywords Xylanase · Bifunctional xylanase/β-glucanase · GH10 · Pretreated Sorghum bicolor bagasse · Antioxidant activity of xylo-oligosaccharides 5 Estación Experimental Agropecuaria Pergamino, Instituto Nacional de Tecnología Agropecuaria (INTA), Pergamino, Buenos Aires, Argentina 6 Instituto de Agrobiotecnología y Biología Molecular (IABIMO), UEDD INTA-CONICET, Hurlingham, Buenos Aires, Argentina Escuela de Ciencias Agrarias y Ambientales-Universidad Nacional del Noroeste de La Provincia de Buenos Aires, Pergamino, Buenos Aires, Argentina 7 Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina Cátedra de Microbiología Agrícola, Facultad de Agronomía, Universidad de Buenos Aires, INBA UBA-CONICET, Ciudad Autónoma de Buenos Aires, Argentina 8 Estación Experimental Agropecuaria Mendoza, Instituto Nacional de Tecnología Agropecuaria (INTA), Luján de Cuyo, Mendoza, Argentina 9 Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina * Ilaria Finore * Paola M. Talia ; 1 2 3 Institute of Biomolecular Chemistry (ICB), Consiglio Nazionale Delle Ricerche (CNR), Pozzuoli, Italy 4 Department of Science and Technology, University of Naples “Parthenope”, Naples, Italy Vol.:(0123456789) 104 Page 2 of 21 Introduction Lignocellulose, which is mainly composed of cellulose, hemicellulose, and lignin, is the most abundant and renewable source of carbon on Earth. However, lignocellulosic biomass from plants is recalcitrant due to its complex polymer composition (Adegboye et al. 2021; Meenakshisundaram et al. 2021). For greater recovery of the sugar components of polysaccharides from lignocellulosic biomass, physical, chemical, and/or biological pretreatment is generally performed before enzymatic hydrolysis. To partially obtain hemicellulose and remove lignin, a basic chemical pretreatment is the most suitable and used method (Basak et al. 2023; Chen et al. 2017; Saini et al. 2015). However, the costs associated with this process are still high, which hinders its large-scale commercialization (Adegboye et al. 2021). Some studies evaluating raw materials for industrial applications have investigated the potency of sweet sorghum (Sorghum bicolor) bagasse (Bagewadi et al. 2016; Castro et al. 2021; Nunta et al. 2023; Wei et al. 2018), which is the residue remaining after extraction of the stem juice and represents approximately 36% of the plant (Pengilly et al. 2015). In addition, sorghum is considered a promising energy crop for use in biofuels and as a source of value-added biomolecules due to its high photosynthetic rate, great genetic diversity, and drought resistance (Barcelos et al. 2016; Bonin et al. 2016). Enzymes, including xylanases and glucanases, play a fundamental role in the transformation of the traditional chemical industry into a more sustainable and environmentally friendly alternative (Talens-Perales et al. 2021). Xylanases (EC 3.2.1.8) catalyze the hydrolysis of the internal 1,4-β-D xylosidic linkages in xylan, the most abundant polysaccharide in hemicellulose in nature. Xylanases occur in several glycosyl hydrolase (GH) families of the Carbohydrate-Active enZYmes (CAZy) database classification, including GH5, GH8, GH10, GH11, GH30, GH43, and GH141. In particular, the GH10 and GH11 families are considered true xylanases because of their substrate specificity (Paës et al. 2012; Talens-Perales et al. 2021). Xylanases are widely used across various industries, including pulp and paper production, juice clarification, enhancement of animal digestibility, and production of second-generation bioethanol (Talens-Perales et al. 2021). More recently, xylanases have been used in the production of xylo-oligosaccharides (XOs), mainly xylobiose to be used as prebiotics, and offers a range of biological benefits, such as antioxidant and antimicrobial effects, among others (Chen et al. 2021; Zarafeta et al. 2020). Glucanases, on the other hand, are able to hydrolyze β-glucan into cello-oligosaccharides (COs) and glucose. Applied Microbiology and Biotechnology (2025) 109:104 They can hydrolyze endo- or exo-β− 1,4-, endo-β− 1,3-, endo-β− 1,3–1,4- or endo-β− 1,3(4)- glycosidic bonds in glucans (Lafond et al. 2012; Lin et al. 2025). Among them, endo-β− 1,4-glucanase (EC 3.2.1.4), exo-glucanase/ cellobiohydrolase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21) are involved in the breakdown of glucan-like substrates, endo-β− 1,3-glucanases or laminarinases (EC 3.2.1.39) hydro (...truncated)