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

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: Antioxidant activity of xylo-oligosaccharides; Bifunctional xylanase/β-glucanase; GH10; Pretreated Sorghum bicolor bagasse; Xylanase.

Fig. 1

Nuclear magnetic resonance spectroscopy (NMR) analysis of polysaccharides recovered after SBB pretreatment; ¹H (upper) (a) and ¹³C (lower) traces were recorded with a Bruker 400 MHz Prodigy instrument (b). The x-axis for each spectrum is expressed as chemical shifts reported as parts per million (ppm) with reference to D₂O and CD₃OD for the ¹H and ¹³C spectra, respectively

Fig. 2

SDS-PAGE analysis of purified Xyl10 C and Xyl10E. Soluble immobilized metal-affinity chromatography (IMAC) purification (12% SDS-PAGE) stained with Coomassie blue. Prestained protein marker (M); total protein content of uninduced culture lysate (WI); total protein content of induced culture lysate (I); first purified elutions (E1) of each xylanase enzyme

Fig. 3

Enzymatic profile activity of Xyl10 C and Xyl10E. Optimal pH condition (a), temperature (b), thermal stability (c), and kinetic analysis (d, e), were evaluated using beechwood xylan, barley β-glucan and pretreated SBB. Concentration of reducing sugars released by hydrolysis of Xyl10 C, Xyl10E, and Xyl10 C + Xyl10E on pretreated SBB (2 h) (f). The results correspond to mean and standard deviations of technical triplicates. Two independent biological replicate assays were performed, with equivalent results. IU, international units: μmol/min

Fig. 4

High-performance liquid chromatography (HPLC) hydrolysis profiles using Xyl10 C, Xyl10E, and their combination on pretreated SBB. Analysis of the xylo-oligosaccharides (XOs): xylobiose (X2), xylotriose (X3), xylotetraose (X4), and xylopentaose (X5) as standards, sample dilution 1:500 (a). Analysis of monosaccharides: arabinose (Ara), galactose (Gal), glucose (Glu), and xylose (X) as standards, sample dilution 1:10 (b)

Fig. 5

Modeled structure of Xyl10 C and Xyl10E. The complete TIM-barrel structures of Xyl10 C (a) and Xyl10E (b) with the catalytic glutamate residues are shown in red. Schematic representation of surface amino acids of Xyl10 C (c), Xyl10B (d), and Xyl10E (e). Red represents the acidic amino acids, whereas light blue represents the surface of the ligand in the catalytic site. Note the differential usage of the acidic amino acids that surround the catalytic site

Fig. 6

Molecular modeling of Xyl10 C and Xyl10E and their interaction with ligands. Schematic 2D Ligplot + representation of non-bonding interactions between the ligand xyloheptaose and Xyl10 C (a) and Xyl10E (b) and between the ligand cellohexaose and Xyl10 C (c) and Xyl10E (d). References in the Ligplot + are as follows: ligand residue names (blue), non-ligand residue names (olive green), H-bond labels (olive green), and hydrophobic residue names (black). References regarding atoms are as follows: nitrogen atoms (blue), oxygen atoms (red), and carbon atoms (black). References regarding bonds are as follows: ligand bonds external bonds (purple), non-ligand bonds (orange), hydrogen bonds (olive green), salt bridges (red), disulphide bonds (gold), external bonds (purple), and hydrophobic “bonds” (brick red). The red circles show the conserved residues in Xyl10 C and Xyl10E, whereas the blue circles indicate the differential amino acids