Search Results (50)

Xylanase, an essential enzyme for breaking down xylan, faces limitations in its industrial applications due to the relatively low catalytic activity of the wild type. Directed evolution was used to enhance the catalytic efficiency of xylanase that originated from the Dickeya dadantii DCE-01. A xylanase variant (Xyn-ep) was obtained with improved catalytic activity by random mutant library employing two rounds of error-prone PCR. The results showed that the Xyn-ep demonstrated enzyme activity 1.6 times higher than that of wild-type xylanase. Sequencing analysis pinpointed key mutation sites at S159P, K212N, and N397S, respectively. Homology modeling was used to analyze the location of the mutation sites and to investigate the mechanism of the improved catalytic performance. The mutant Xyn-ep showed improved catalytic performance by error-prone PCR. Additionally, the increased flexibility of the loop of the mutant may contribute to the enhanced activity. These findings indicate that error-prone PCR is an effective method for enhancing enzyme activity and that the mutant Xyn-ep may be a new GH30 xylanase, being a potential candidate for industrial applications such as bast fiber bio-degumming, cotton bio-refinery, paper making, and so on. Full article

Protein packing within crystal lattices plays a critical role in determining molecular flexibility; therefore, the observed conformation and flexibility of protein side chains can vary depending on the crystal space group. Protein crystal dehydration affects crystal lattice mosaicity, which can either reduce crystal quality or enhance X-ray diffraction intensity. It also often alters the crystal lattice, leading to space group transition. Accordingly, dehydration-induced space group transitions could theoretically offer an alternative when there are experimental limitations obstructing the obtainment of diverse crystal forms. However, this remains underexplored experimentally. Here, a dehydration-induced space group transition was explored to observe different conformations and flexibilities of the protein structure. Xylanase GH11 crystals from Thermoanaerobacterium saccharolyticum (TsaGH11) were air-dehydrated, and their structure at room temperature was determined. Upon dehydration, the space group of the TsaGH11 crystal changed from tetragonal to orthorhombic, affecting the protein–protein interfaces within the crystal lattice. The dehydrated crystal structure of TsaGH11 revealed multiple conformations of residues involved in substrate binding and recognition within the substrate-binding cleft. These diverse molecular conformations and flexibilities provide significant and previously unrevealed structural information for TsaGH11. This approach demonstrates the potential of dehydration-induced space group transitions to reveal diverse protein conformations, offering valuable insights into molecular properties and functions. Full article

Thermostable cellulases and xylanases have broad acceptance in food, feed, paper and pulp, and bioconversion of lignocellulosics. Thermophilic fungi serve as an excellent source of thermostable enzymes. This study characterized four endo-β-1,4-glucanases (two glycoside hydrolase (GH) family 5 and two GH7 members) and four endo-β-1,4-xylanases (two GH10 and two GH11 members) from thermophilic fungus Thielavia terrestris, along with one GH10 endo-β-1,4-xylanase each from thermophilic fungus Chaetomium thermophilum and mesophilic fungus Chaetomium globosum. Comparative analysis was conducted against three previously reported GH10 endoxylanases: two thermostable enzymes from the thermophilic fungus Humicola insolens and thermophilic bacterium Halalkalibacterium halodurans, and one mesophilic enzyme from model fungus Neurospora crassa. The GH10 xylanase TtXyn10C (Thite_2118148; UniProt G2R8T7) from T. terrestris demonstrated high thermostability and activity, with an optimal temperature of 80–85 °C. It retained over 60% of its activity after 2 h at 70 °C, maintained approximately 30% activity after 15 min at 80 °C, and showed nearly complete stability following 1 min of exposure to 95 °C. TtXyn10C exhibited specific activity toward beechwood xylan (1130 ± 15 U/mg) that exceeded xylanases from H. insolens and H. halodurans while being comparable to N. crassa xylanase activity. Furthermore, TtXyn10C maintained stability across a pH range of 3–9 and resisted trypsin digestion, indicating its broad applicability. The study expands understanding of enzymes from thermophilic fungi. The discovery of the TtXyn10C offers a new model for investigating the high activity-stability trade-off and structure-activity relationships critical for industrial enzymes. Full article

Rumen microbiota is crucial for cellulose degradation and nutrient metabolism in ruminants. Different feeding systems like grazing and housed feeding can significantly impact it. Mongolian cattle show unique cellulose degradation ability, but functional changes under different feeding conditions are unclear. This study aims to investigate the effects of grazing and housed feeding on rumen microbiota, cellulose degradation, and metabolism in Mongolian cattle. In a 90-day trial, 12 female Mongolian cattle were divided into grazing (F group) and housed feeding (S group). Rumen samples were collected to analyze fermentation parameters, enzyme activities, microbiomes, and metabolomes. The F group had higher acetate, cellulase, xylanase, and β-glucosidase activities (p < 0.05). Bacteroidota and Prevotella were more abundant (p < 0.05), while Firmicutes and Ruminococcus were less abundant (p < 0.05) in the F group. Carbohydrate metabolic pathways and CAZymes (GH2, GH10) were upregulated in the F group, while the S group had enriched purine metabolic pathways and CAZyme (GH31). A total of 64 differential metabolites were found, with subaphylline upregulated in the F group and L-arogenate in the S group (p < 0.05). Grazing increased cellulose degradation and subaphylline production in Mongolian cattle, while housed feeding improved starch utilization efficiency and fat synthesis. These findings provide a basis for optimizing feeding strategies and improving fibrous feed resource utilization in Mongolian cattle. Full article

Xylanases, important enzymes in the food industry, have severely limited use in industrial applications due to insufficient thermal stability. This study focused on improving the thermostability of XynASP, a glycoside hydrolase family 11 (GH11) xylanase from Aspergillus saccharolyticus JOP 1030-1, through modular assembly and rational mutagenesis. By aligning XynASP with nine thermostable GH11 homologs, six variable structural modules (β1, β3, β6, β7, α1, β14) and eight non-conserved residues were identified. Six chimeras (Z1, Z2, Z3, Z4, Z5, Z6) and eight single mutants (S131T, Y133T, A137G, A144T, T147Y, A156R, V198M, and Y204Q) were constructed. Among these, the β3-module-substituted chimera Z2 exhibited a 15.4-fold extended half-life at 45 °C compared to wild-type XynASP. Single-point mutagenesis revealed that V198M showed the highest residual activity after thermal treatment. To further optimize stability, combinatorial mutagenesis was performed: the double mutant A144T/V198M demonstrated a 4.3-fold longer half-life at 50 °C. Combining Z2 with the A144T/V198M mutations yielded the chimeric ZM, which demonstrated a 26.5-fold increase in half-life at 50 °C and a 5.5-fold improvement in catalytic efficiency (197.4 U/mg) compared to wild-type XynASP. Structural analysis and molecular dynamics simulations showed that increased hydrophobic interactions at both the N- and C-termini improved the structural stability of chimeric ZM, while increasing the flexibility of the thumb can offset the negative impact on catalytic activity during thermal stability modification of GH11 xylanase. This study further confirmed that modular assembly is an effective approach for obtaining high-activity, heat-resistant xylanases. This study also notably deepened our understanding of the thermal stability mechanisms of xylanases. Full article

This study investigates the biochemical properties of two xylanases, ZgXyn10A and CaXyn10B, which are members of the glycoside hydrolase family 10 (GH10) and originate from the marine Bacteroidetes species Zobellia galactanivorans and Cellulophaga algicola, respectively. Utilizing an auto-induction expression system in Escherichia coli, high-purity recombinant forms of these enzymes were successfully produced. Biochemical assays revealed that ZgXyn10A and CaXyn10B exhibit optimal activities at 40 °C and 30 °C, respectively, and demonstrate a high sensitivity to temperature fluctuations. Unlike conventional low-temperature enzymes, these xylanases retain only a fraction of their maximal activity at lower temperatures. To gain deeper insights into the structural and functional properties of these marine xylanases, two thermostable GH10 xylanases, TmxB and CoXyn10A, which share comparable amino acid sequence identity with ZgXyn10A and CaXyn10B, were selected for structural comparison. All four marine xylanases share a nearly similar three-dimensional structural topology. Molecular dynamics simulation indicated a striking difference in structural fluctuations between the low-temperature and thermostable xylanases, as evidenced by the distinct root mean square deviation values. Moreover, root mean square fluctuation analysis specifically identified the β3-α3 and β7-α7 loop regions within the substrate-binding cleft as crucial determinants of the temperature characteristics of these GH10 xylanases. Our findings establish loop dynamics as a key evolutionary driver in the thermal adaptation of GH10 xylanases and propose a loop engineering strategy for the development of industrial biocatalysts with tailored temperature responses, particularly for lignocellulosic biomass processing under moderate thermal conditions. Full article

Glycoside hydrolase family 11 (GH11) xylanases are used in various industries, such as biorefining, animal feed production, and baking, making them key industrial enzymes. Operating bioprocesses at elevated temperatures enhances the reaction rate and product yield and thus requires thermostable enzymes to sustain catalytic performance. The limited availability of naturally occurring thermostable GH11 xylanases necessitates targeted modifications via protein engineering to enhance their thermal stability. In this review, we present the key drivers of thermostability, an overview of engineering strategies, and the underlying mechanisms of action. Finally, we investigated state-of-the-art technologies involving artificial intelligence (AI)- and ancestral sequence reconstruction-guided approaches. Full article

Xylanases are crucial for the breakdown of hemicellulose, enabling the conversion of lignocellulosic biomass into fermentable sugars for biofuels and other industrial applications. For the first time, we investigated the biochemical and genetic characteristics of 22 xylanase genes from Thermothelomyces fergusii within glycoside hydrolase (GH) families GH10, GH11, and GH43. Xylanase genes structural diversity clustered the phylogenetic tree into GH10, GH11, GH43-I, and GH43-II groups. Structural analysis revealed that all TfGH10 and TfGH11 genes contained conserved GH domains, with CBM1 present in TfGH10-5 and TfGH11-4. Secondary domains, including CBM35, CBM42, and CBM91, were found in the GH43 gene family. The presence of key glutamic (Glu) and aspartic (Asp) residues in active sites is essential for substrate binding and catalysis. RT-qPCR analysis revealed substrate-dependent gene expression, with peak upregulation on day three in beechwood xylan (BWX) cultures and day two in corncob xylan (CCX) and rice straw (RS) cultures. Consistent with these findings, enzymatic assays demonstrated the highest xylanase activity in BWX-induced cultures, followed by RS and CCX, underscoring the differential regulation of these enzymes in response to distinct hemicellulosic substrates. These findings provide valuable insights into the structural, functional, and regulatory mechanisms of T. fergusii xylanases, facilitating their industrial application. Full article

GH10 xylanases and GH62 Arabinofuranosidases play key roles in the breakdown of arabinoxylans and are important tools in various industrial and biotechnological processes, such as renewable biofuel production, the paper industry, and the production of short-chain xylooligosaccharides (XOS) from plant biomass. However, the use of these enzymes in industrial settings is often limited due to their relatively low thermostability and reduced catalytic efficiency. To overcome these limitations, strategies based on enzymatic chimera construction and the use of metal ions and other cofactors have been proposed to produce new recombinant enzymes with improved catalytic activity and thermostability. Here, we examine the conformational dynamics of a GH10-GH62 chimera at different calcium ion concentrations through molecular dynamics simulations. While experimental data have demonstrated improved activity and thermostability in GH10-GH62 chimera, the mechanistic basis for these enhancements remains unclear. We explored the structural details of the binding subsites of Ca²⁺ in the parental enzymes GH62 from Aspergillus fumigatus (Afafu62) and a recombinant GH10 from Cryptococcus flavescens (Xyn10cf), as well as their chimeric combination, and how negatively charged electron pairing located at the protein surface affects Ca²⁺ capture. The results indicate that Ca²⁺ binding significantly contributes to structural stability and catalytic cavity modulation in the chimera, particularly evident at a concentration of 0.01 M. This effect, not observed in the parental GH10 and GH62 enzymes, highlights how Ca²⁺ enhances stability in the overall chimeric enzyme, while supporting a larger cavity volume in the chimera GH62 subunit. The increased catalytic site volume and reduced structural flexibility in response to Ca²⁺ suggest that calcium binding minimizes non-productive conformational states, which could potentially improve catalytic turnover. The findings presented here may aid in the development of more thermostable and efficient catalytic systems. Full article

This study identified a salt-tolerant GH11 xylanase, Xyn^st, which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xyn^st and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xyn^st in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan (K_m = 0.30 mg mL⁻¹ for Xyn^st and 0.18 mg mL⁻¹ for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (K_cat/K_m = 15483.33 mL mg⁻¹ s⁻¹). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xyn^st did. The conversion efficiencies of Xyn^st and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries. Full article

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-β-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-β-xylanase NhX1 showed maximum activity and stability at pH 6.0–7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed K_m 2.16 mg/mL and V_max 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-β-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria. Full article

Rising temperature is a major threat to the normal growth and development of maize, resulting in low yield production and quality. The mechanism of maize in response to heat stress remains uncertain. In this study, a maize mutant Zmhsl-1 (heat sensitive leaves) with wilting and curling leaves under high temperatures was identified from maize Zheng 58 (Z58) mutant lines generated by ethyl methanesulfonate (EMS) mutagenesis. The Zmhsl-1 plants were more sensitive to increased temperature than Z58 in the field during growth season. The Zmhsl-1 plants had lower plant height, lower yield, and lower content of photosynthetic pigments. A bulked segregant analysis coupled with whole-genome sequencing (BSA-seq) enabled the identification of the corresponding gene, named ZmHSL, which encodes an endo-β-1,4-xylanase from the GH10 family. The loss-of-function of ZmHSL resulted in reduced lignin content in Zmhsl-1 plants, leading to defects in water transport and more severe leaf wilting with the increase in temperature. RNA-seq analysis revealed that the differentially expressed genes identified between Z58 and Zmhsl-1 plants are mainly related to heat stress-responsive genes and unfolded protein response genes. All these data indicated that ZmHSL plays a key role in lignin synthesis, and its defective mutation causes changes in the cell wall structure and gene expression patterns, which impedes water transport and confers higher sensitivity to high-temperature stress. Full article

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. Full article

The endo-β-1,4-xylanase glycosyl hydrolase (GH11) decomposes the backbone of xylan into xylooligosaccharides or xylose. These enzymes are important for industrial applications in the production of biofuel, feed, food, and value-added materials. β-D-xylopyranose (XYP, also known as β-D-xylose) is the fundamental unit of the substrate xylan, and understanding its recognition is fundamental for the initial steps of GH11’s molecular mechanism. However, little is known about the recognition of a single XYP molecule by GH11. In this study, the crystal structures of GH11 from Thermoanaerobacterium saccharolyticum (TsaGH11) complexed with an XYP molecule were determined at a resolution of 1.7–1.9 Å. The XYP molecule binds to subsite −2 of the substrate-binding cleft. The XYP molecule is mainly stabilized by a π–π interaction with the conserved Trp36 residue. The O2 and O3 atoms of XYP are stabilized by hydrogen bond interactions with the hydroxyl groups of Tyr96 and Tyr192. The conformation of the thumb domain of TsaGH11 does not play a critical role in XYP binding, and XYP binding induces a shift in the thumb domain of TsaGH11 toward the XYP molecule. A structural comparison of TsaGH11 with other GH11 xylanases revealed that the XYP molecule forms π–π stacking with the center between the phenyl and indoline ring of Trp36, whereas the XYP molecule unit from xylobiose or xylotetraose forms π–π stacking with the indoline of Trp36, which indicates that the binding modes of the substrate and XYP differ. These structural results provide a greater understanding of the recognition of XYP by the GH11 family. Full article

Proteins can form crystals spontaneously without crystallization experiments. These crystals can be used to determine three-dimensional structures. However, when X-ray diffraction is poor, crystal optimization is required to obtain a high-resolution crystal structure. Endo-1,4-β-xylanase from the fungus Hypocrea virens (HviGH11) spontaneously formed microcrystals after affinity purification and concentration; however, most HviGH11 microcrystals showed poor diffraction in the synchrotron X-ray and X-ray free-electron laser, so a complete three-dimensional structure could not be obtained. This study presents a method to improve the crystal quality of spontaneously grown HviGH11 microcrystals. The crystallization screening results revealed that temperature, pH, and salt were not crucial factors in increasing the solubility or preventing the spontaneous crystal growth of HviGH11. Conversely, the addition of polyethylene glycols (PEGs) as a precipitant facilitated the growth of larger HviGH11 crystals. The improved large HviGH11 crystal showed a diffraction of up to 1.95 Å when exposed to synchrotron X-rays, providing a complete three-dimensional structural dataset. Based on the nucleation rate equation, it was suggested that PEG increases the viscosity of the protein solution rather than promoting nucleation. This increase in viscosity reduced nucleation and facilitated the growth of larger HviGH11 crystals. These results provide valuable insights for future experiments aimed at increasing the size of spontaneously grown crystals. Full article