Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase.

Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite -2. Furthermore, the protein has a larger ß5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase.

Molecular mechanism of cellulose depolymerization by the two-domain BlCel9A enzyme from the glycoside hydrolase family 9.

Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs.

Paludibacter propionicigenes GH10 xylanase as a tool for enzymatic xylooligosaccharides production from heteroxylans.

Bioconversion of lignocellulosic biomass into value-added products relies on polysaccharides depolymerization by carbohydrate active enzymes. This work reports biochemical characterization of Paludibacter propionicigenes xylanase from GH10 (PpXyn10A) and its application for enzymatic xylooligosaccharides (XOS) production from commercial heteroxylans and liquor of hydrothermally pretreated corn cobs (PCC). PpXyn10A is tolerant to ethanol and NaCl, and releases xylobiose (X2) and xylotriose (X3) as the main hydrolytic products. The conversion rate of complex substrates into short XOS was approximately 30% for glucuronoxylan and 8.8% for rye arabinoxylan, after only 4 h; while for PCC, PpXyn10A greatly increased unbranched XOS yields. B. adolescentis fermentation with XOS from beechwood glucuronoxylan produced mainly acetic and lactic acids. Structural analysis shows that while the glycone region of PpXyn10A active site is well preserved, the aglycone region has aromatic interactions in the +2 subsite that may explain why PpXyn10A does not release xylose.

Recent advances in the enzymatic production and applications of xylooligosaccharides.

The majority of lignocellulosic biomass on the planet originates from plant cell walls, which are complex structures build up mainly by cellulose, hemicellulose and lignin. The largest part of hemicellulose, xylan, is a polymer with a ß-(1→4)-linked xylose residues backbone decorated with α-D-glucopyranosyl uronic acids and/or L-arabinofuranose residues. Xylan is the second most abundant biopolymer in nature, which can be sustainably and efficiently degraded into decorated and undecorated xylooligosaccharides (XOS) using combinations of thermochemical pretreatments and enzymatic hydrolyses, that have broad applications in the food, feed, pharmaceutical and cosmetic industries. Endo-xylanases from different complex carbohydrate-active enzyme (CAZyme) families can be used to cleave the backbone of arabino(glucurono)xylans and xylooligosaccharides and degrade them into short XOS. It has been shown that XOS with a low degree of polymerization have enhanced prebiotic effects conferring health benefits to humans and animals. In this review we describe recent advances in the enzymatic production of XOS from lignocellulosic biomass arabino- and glucuronoxylans and their applications as food and feed additives and health-promoting ingredients. Comparative advantages of xylanases from different CAZy families in XOS production are discussed and potential health benefits of different XOS are presented.

Production of prebiotic xylooligosaccharides from arabino- and glucuronoxylan using a two-domain Jonesia denitrificans xylanase from GH10 family.

Development of a more environmentally sustainable society is based on the maximum use of renewable carbon sources and their valorization of environmentally-friendly green technologies. This includes a thorough use of plant biomass and agricultural residues for the production of value-added bioproducts. Xylan is the second most abundant biopolymer in nature which can be sustainable converted into pentoses and xylooligosaccharides, that have wide applications in the food, feed, pharmaceutical, and cosmetic industry. Within the scope of present study, we biochemically characterized two-domain GH10 xylanase from Jonesia denitrificans (JdXyn10A) and evaluated its applicability for production of xylooligosaccharides (XOS). JdXyn10A has a specific activity of 84 ± 2 U/mg and 65 ± 5 U/mg when acting on beechwood glucuronoxylan and rye arabinoxylan, respectively. The enzyme is stable in a wide pH range and is tolerant to high concentrations of NaCl and ethanol. Interestingly, the profile of products released by the enzyme is predominant in xylobiose and xylotriose, with a very low fraction of xylose which is desirable for XOS production. The efficiencies of enzymatic conversion of beechwood glucuronoxylan and rye arabinoxylan are 47.67 % and 26.01 %, respectively, after 6 h of enzymatic hydrolysis only. Structural comparison between the JdXyn10A homology model and the structure from its homologous that while the glycone region of its active site is well preserved, the aglycone region presents structural differences in the +2 subsite that may explain why JdXyn10A does not release xylose.