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Introduction: Verticillium dahliae causes a devastating Verticillium wilt disease on hundreds of plant species worldwide, including cotton. Understanding the interaction mechanism between V. dahliae and its hosts is the prerequisite for developing effective strategies for disease prevention. Methods: Here, based on the previous observation of an xylosidase-encoding gene (VdxyL3) in V. dahliae being obviously up-regulated after sensing root exudates from a cotton variety susceptible to this pathogen, we investigated the function of VdxyL3 in the growth and pathogenesis of V. dahliae by generating its deletion-mutant strains (1VdxyL3). Results: Deleting VdxyL3 led to increased colony expansion rate, conidial production, mycelial growth, carbon and nitrogen utilization capacities, and enhanced stress tolerance and pathogenicity of V. dahliae. VdxyL3 is a secretory protein; however, VdxyL3 failed to induce cell death in N. benthamiana based on transient expression experiment. Transcriptomic analysis identified 1300 genes differentially expressed (DEGs) between wild-type (Vd952) and 1VdxyL3 during infection, including 348 DEGs encoding secretory proteins, among which contained 122 classical secreted proteins and 226 non-classical secreted proteins. It was notable that of the 122 classical secretory proteins, 50 were carbohydrate-active enzymes (CAZymes) and 58 were small cysteine rich proteins (SCRPs), which were required for the pathogenicity of V. dahliae. Conclusion: The RNA-seq data thus potentially connected the genes encoding these proteins to the pathogenesis of V. dahliae. This study provides an experimental basis for further studies on the interaction between V. dahliae and cotton and the pathogenic mechanism of the fungus. [ABSTRACT FROM AUTHOR]

Abstract Objective New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) β-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase. Results In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of β-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists.

Objective: New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) β-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase. Results: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of β-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists. [ABSTRACT FROM AUTHOR]

The study of soil microbial biomass and enzymatic activity of natural and anthropogenically transformed ecosystems was carried out. The catenas of virgin luvisols and chernozems of the Belogorye Natural Reserve and the catenas of arable soils were studied under similar geomorphological and lithological conditions. The activities of enzymes involved in the cycles of carbon (β-glucosidase and xylosidase), nitrogen (chitinase), and phosphorus (acid and alkaline phosphatase) was studied. It has been established that a decrease in soil microbial biomass as a result of plowing is not accompanied by an equivalent decrease in the enzymatic activity of the soil. Differences in the enzymatic activity of different soils types were revealed, which indicates differences in the structure of the microbial community and the type of phytocenoses. Patterns of changes in the enzymatic activity of soils in watershed areas, in the transit and accumulative parts of catenas, have been established. The values of specific activities of enzymes (enzymatic activities per unit of microbial biomass) were calculated. The obtained patterns of changes in the specific enzymatic activity of arable soils indicate that, despite the loss of organic matter and a decrease in microbial biomass as a result of plowing, the physiological efficiency of the microbial community of agrochernozem is higher than in virgin soil. High specific enzymatic activity in arable soils is associated with higher rate of enzyme production by soil microorganisms due to land use changes. [ABSTRACT FROM AUTHOR]

Plant cell wall-derived oligosaccharides, i.e., damage-associated molecular patterns (DAMPs), could be generated after pathogen attack or during normal plant development, perceived by cell wall receptors, and can alter immunity and cell wall composition. Therefore, we hypothesised that xylo-oligosaccharides (XOS) could act as an elicitor and trigger immune responses. To test this, we treated Arabidopsis with xylobiose (XB) and investigated different parameters. XB-treatment significantly triggered the generation of reactive oxygen species (ROS), activated MAPK protein phosphorylation, and induced callose deposition. The combination of XB (DAMP) and flg22 a microbe-associated molecular pattern (MAMP) further enhanced ROS response and gene expression of PTI marker genes. RNA sequencing analysis revealed that more genes were differentially regulated after 30 min compared to 24 h XB-treated leaves, which correlated with ROS response. Increased xylosidase activity and soluble xylose level after 30 min and 3 h of XB-treatment were observed which might have weakened the DAMP response. However, an increase in total cell wall sugar and a decrease in uronic acid level was observed at both 30 min and 24 h. Additionally, arabinose, rhamnose, and xylose levels were increased in 30 min, and glucose was increased in 24 h compared to mock-treated leaves. The level of jasmonic acid, abscisic acid, auxin, and cytokinin were also affected after XB treatment. Overall, our data revealed that the shortest XOS can act as a DAMP, which triggers the PTI response and alters cell wall composition and hormone level. Key message: Xylobiose treatment in Arabidopsis rosette leaf induces plant-triggered immunity (PTI) responses, alters cell wall polysaccharide composition and influences growth hormone levels. [ABSTRACT FROM AUTHOR]

Xylanase and β-xylosidase are the key enzymes for hemicellulose hydrolysis. To further improve hydrolysis efficacy, high temperature hydrolysis with thermostable hemicellulases showed promise. In this study, thermostable xylanase (Xyn) and β-xylosidase (XynB) genes from Pseudothermotoga thermarum were cloned and secretory expressed in Bacillu subtilis. Compared with Escherichia coli expression host, B. subtilis resulted in a 1.5 time increase of enzymatic activity for both recombinant enzymes. The optimal temperature and pH were 95°C and 6.5 for Xyn, and 95°C and 6.0 for XynB. Thermostability of both recombinant enzymes was observed between the temperature range of 75–85°C. Molecular docking analysis through AutoDock showed the involvement of Glu525, Asn526, Trp774 and Arg784 in Xyn-ligand interaction, and Val237, Lys238, Val761 and Asn76 in XynB-ligand interaction, respectively. The recombinant Xyn and XynB exhibited synergistic hydrolysis of beechwood xylan and pretreated lignocellulose, where Xyn and XynB pre-hydrolysis achieved a better improvement of pretreated lignocellulose hydrolysis by commercial cellulase. The observed stability of the enzymes at high temperature and the synergistic effect on lignocellulosic substrates suggested possible application of these enzymes in the field of saccharification process.

Pectin- and hemicellulose-associated structures of plant cell walls participate in defense responses against pathogens of different parasitic lifestyles. The resulting immune responses incorporate phytohormone signaling components associated with salicylic acid (SA) and jasmonic acid (JA). SA plays a pivotal role in systemic acquired resistance (SAR), a form of induced resistance that - after a local immune stimulus - confers long-lasting, systemic protection against a broad range of biotrophic invaders. β-D-XYLOSIDASE 4 (BXL4) protein accumulation is enhanced in the apoplast of plants undergoing SAR. Here, two independent Arabidopsis thaliana mutants of BXL4 displayed compromised systemic defenses, while local resistance responses to Pseudomonas syringae remained largely intact. Because both phloem-mediated and airborne systemic signaling were abrogated in the mutants, the data suggest that BXL4 is a central component in SAR signaling mechanisms. Exogenous xylose, a possible product of BXL4 enzymatic activity in plant cell walls, enhanced systemic defenses. However, GC-MS analysis of SAR-activated plants revealed BXL4-associated changes in the accumulation of certain amino acids and soluble sugars, but not xylose. In contrast, the data suggest a possible role of pectin-associated fucose as well as of the polyamine putrescine as regulatory components of SAR. This is the first evidence of a central role of cell wall metabolic changes in systemic immunity. Additionally, the data reveal a so far unrecognized complexity in the regulation of SAR, which might allow the design of (crop) plant protection measures including SAR-associated cell wall components. [ABSTRACT FROM AUTHOR]

Commercially available cellulases and amylases can disperse the pathogenic bacteria embedded in biofilms. This suggests that polysaccharide-degrading enzymes would be useful as antibacterial therapies to aid the treatment of biofilm-associated bacteria, e.g., in chronic wounds. Using a published enzyme library, we explored the capacity of 76 diverse recombinant glycoside hydrolases to disperse Staphylococcus aureus biofilms. Four of the 76 recombinant glycoside hydrolases digested purified cellulose, amylose, or pectin. However, these enzymes did not disperse biofilms, indicating that anti-biofilm activity is not general to all glycoside hydrolases and that biofilm activity cannot be predicted from the activity on pure substrates. Only one of the 76 recombinant enzymes was detectably active in biofilm dispersion, an α-xylosidase from Aspergillus nidulans. An α-xylosidase cloned subsequently from Aspergillus thermomutatus likewise demonstrated antibiofilm activity, suggesting that α-xylosidases, in general, can disperse Staphylococcus biofilms. Surprisingly, neither of the two β-xylosidases in the library degraded biofilms. Commercial preparations of amylase and cellulase that are known to be effective in the dispersion of Staphylococcus biofilms were also analyzed. The commercial cellulase contained contaminating proteins with multiple enzymes exhibiting biofilm-dispersing activity. Successfully prospecting for additional antibiofilm enzymes may thus require large libraries and may benefit from purified enzymes. The complexity of biofilms and the diversity of glycoside hydrolases continue to make it difficult to predict or understand the enzymes that could have future therapeutic applications. [ABSTRACT FROM AUTHOR]

Pectin- and hemicellulose-associated structures of plant cell walls participate in defense responses against pathogens of different parasitic lifestyles. The resulting immune responses incorporate phytohormone signaling components associated with salicylic acid (SA) and jasmonic acid (JA). SA plays a pivotal role in systemic acquired resistance (SAR), a form of induced resistance that - after a local immune stimulus - confers long-lasting, systemic protection against a broad range of biotrophic invaders. β-D-XYLOSIDASE 4 (BXL4) protein accumulation is enhanced in the apoplast of plants undergoing SAR. Here, two independent Arabidopsis thaliana mutants of BXL4 displayed compromised systemic defenses, while local resistance responses to Pseudomonas syringae remained largely intact. Because both phloem-mediated and airborne systemic signaling were abrogated in the mutants, the data suggest that BXL4 is a central component in SAR signaling mechanisms. Exogenous xylose, a possible product of BXL4 enzymatic activity in plant cell walls, enhanced systemic defenses. However, GC-MS analysis of SAR-activated plants revealed BXL4-associated changes in the accumulation of certain amino acids and soluble sugars, but not xylose. In contrast, the data suggest a possible role of pectin-associated fucose as well as of the polyamine putrescine as regulatory components of SAR. This is the first evidence of a central role of cell wall metabolic changes in systemic immunity. Additionally, the data reveal a so far unrecognized complexity in the regulation of SAR, which might allow the design of (crop) plant protection measures including SAR-associated cell wall components.

Development of chimeric enzymes by protein engineering can more efficiently contribute toward biomass conversion for bioenergy generation. Therefore, prior to experimental validation, a computational approach by modeling and molecular dynamic simulation can assess the structural and functional behavior of chimeric enzymes. In this study, a bifunctional chimera, CtXyn11A-BoGH43A comprising an efficient endoxylanase (CtXyn11A) from Clostridium thermocellum and xylosidase (BoGH43A) from Bacteroides ovatus was computationally designed and its binding and stability analysis with xylooligosaccharides were performed. The modeled chimera showed β-jellyroll fold for CtXyn11A and 5-bladed β-propeller fold for BoGH43A module. Stereo-chemical properties analyzed by Ramachandran plot showed 98.8% residues in allowed region, validating the modeled chimera. The catalytic residues identified by multiple sequence alignment were Glu94 and Glu184 for CtXyn11A and Asp229 and Glu384 for BoGH43A modules. CtXyn11A followed retaining-type, whereas BoGH43A enforced inverting-type of reaction mechanism during xylan hydrolysis as revealed by superposition and GH11 and GH43 familial analyses. Molecular docking studies showed binding energy, (ΔG) − 4.54 and − 4.18 kcal/mol for CtXyn11A and BoGH43A modules of chimera, respectively, with xylobiose, while − 3.94 and − 3.82 kcal/mol for CtXyn11A and BoGH43A modules of chimera, respectively, with xylotriose. MD simulation of CtXyn11A-BoGH43A complexed with xylobiose and xylotriose till 100 ns displayed stability by RMSD, compactness by Rg and conformational stability by SASA analyses. The lowered values of RMSF in active-site residues, Glu94, Glu184, Asp229, Asp335 and Glu384 confirmed the efficient binding of chimera with xylobiose and xylotriose. These results were in agreement with the earlier experimental studies on CtXyn11A releasing xylooligosaccharides from xylan and BoGH43A releasing d-xylose from xylooligosaccharides and xylobiose. The chimera showed stronger affinity in terms of total short-range interaction energy; − 190 and − 121 kJ/mol for with xylobiose and xylotriose, respectively. The bifunctional chimera, CtXyn11A-BoGH43A showed stability and integrity with xylobiose and xylotriose. The designed chimera can be constructed and applied for efficient biomass conversion. [ABSTRACT FROM AUTHOR]

Xylosidases are widely used for the production of prebiotics and the transformation of natural active substances in the food industry. However, xylosidases with excellent thermostability and product tolerance are required for industrial applications. In this study, the thermostability and final-product tolerance of the previously reported robust xylosidase Xyl21 were further improved via directed evolution. The triple mutant variant Xyl21-A16 (K16R, L94I, and K262N) showed significantly enhanced xylose tolerance, ethanol tolerance, and thermostability with no apparent changes in the specific activity, optimum pH, and temperature compared with the wild type. Single site mutations suggested that variant Xyl21-A16 is the cumulative result of three mutated sites, which indicated that K16 and L94 play important roles in enzyme characteristics. Moreover, a comparison of the predicted protein structures of Xyl21 and its variant indicated that additional molecular interactions formed by K16R and K262N might directly improve the rigidity of the protein structure, therefore contributing to the increased thermostability and product tolerance. The variant Xyl21-A16 developed in this study has great application potential in the production of prebiotics, and also provides a useful reference for the future engineering of other xylosidases. [ABSTRACT FROM AUTHOR]

Improving the specific activity and thermostability of psychrophilic xylosidase is important for improving its enzymatic performance and promoting its industrial application. Herein, a psychrophilic xylosidase AX543 exhibited activity in the temperature range between 0 and 35 °C, with optimum activity at 20 °C, which is lower than that of other reported psychrophilic xylosidases. The thermostability, specific activity, and catalytic efficiency of the site-directed variants G110S, Q201R, and L2 were significantly enhanced, without affecting the optimal reaction temperature. Comparative protein structural analysis and molecular dynamics simulation indicated that these improvements might be the result of the increased hydrogen bonds interaction and improved structural rigidity. Furthermore, homologous module substitution with four segments demonstrated that the psychrophilic characteristics of AX543 are the results of the whole protein structure, and the C-terminal segment A4 appears to be more essential in determining psychrophilic characteristics, exhibiting potentiality to produce more psychrophilic xylosidases. This study provides valuable structural information on psychrophilic xylosidases and also offers attractive modification strategies to modify catalytic activity, thermostability, and optimal reaction temperature. [ABSTRACT FROM AUTHOR]

Commercially available cellulases and amylases can disperse the pathogenic bacteria embedded in biofilms. This suggests that polysaccharide-degrading enzymes would be useful as antibacterial therapies to aid the treatment of biofilm-associated bacteria, e.g., in chronic wounds. Using a published enzyme library, we explored the capacity of 76 diverse recombinant glycoside hydrolases to disperse Staphylococcus aureus biofilms. Four of the 76 recombinant glycoside hydrolases digested purified cellulose, amylose, or pectin. However, these enzymes did not disperse biofilms, indicating that anti-biofilm activity is not general to all glycoside hydrolases and that biofilm activity cannot be predicted from the activity on pure substrates. Only one of the 76 recombinant enzymes was detectably active in biofilm dispersion, an α-xylosidase from Aspergillus nidulans. An α-xylosidase cloned subsequently from Aspergillus thermomutatus likewise demonstrated antibiofilm activity, suggesting that α-xylosidases, in general, can disperse Staphylococcus biofilms. Surprisingly, neither of the two β-xylosidases in the library degraded biofilms. Commercial preparations of amylase and cellulase that are known to be effective in the dispersion of Staphylococcus biofilms were also analyzed. The commercial cellulase contained contaminating proteins with multiple enzymes exhibiting biofilm-dispersing activity. Successfully prospecting for additional antibiofilm enzymes may thus require large libraries and may benefit from purified enzymes. The complexity of biofilms and the diversity of glycoside hydrolases continue to make it difficult to predict or understand the enzymes that could have future therapeutic applications.

Introduction: Verticillium dahliae causes a devastating Verticillium wilt disease on hundreds of plant species worldwide, including cotton. Understanding the interaction mechanism between V. dahliae and its hosts is the prerequisite for developing effective strategies for disease prevention., Methods: Here, based on the previous observation of an xylosidase-encoding gene ( VdxyL3 ) in V. dahliae being obviously up-regulated after sensing root exudates from a cotton variety susceptible to this pathogen, we investigated the function of VdxyL3 in the growth and pathogenesis of V. dahliae by generating its deletion-mutant strains ( ΔVdxyL3 )., Results: Deleting VdxyL3 led to increased colony expansion rate, conidial production, mycelial growth, carbon and nitrogen utilization capacities, and enhanced stress tolerance and pathogenicity of V. dahliae . VdxyL3 is a secretory protein; however, VdxyL3 failed to induce cell death in N. benthamiana based on transient expression experiment. Transcriptomic analysis identified 1300 genes differentially expressed (DEGs) between wild-type (Vd952) and ΔVdxyL3 during infection, including 348 DEGs encoding secretory proteins, among which contained 122 classical secreted proteins and 226 non-classical secreted proteins. It was notable that of the 122 classical secretory proteins, 50 were carbohydrate-active enzymes (CAZymes) and 58 were small cysteine rich proteins (SCRPs), which were required for the pathogenicity of V. dahliae ., Conclusion: The RNA-seq data thus potentially connected the genes encoding these proteins to the pathogenesis of V. dahliae . This study provides an experimental basis for further studies on the interaction between V. dahliae and cotton and the pathogenic mechanism of the fungus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Song, Chen, Zhang, Sun, Ma, Li, Sun and Zhang.)

Xylosidases are widely used for the production of prebiotics and the transformation of natural active substances in the food industry. However, xylosidases with excellent thermostability and product tolerance are required for industrial applications. In this study, the thermostability and final-product tolerance of the previously reported robust xylosidase Xyl21 were further improved via directed evolution. The triple mutant variant Xyl21-A16 (K16R, L94I, and K262N) showed significantly enhanced xylose tolerance, ethanol tolerance, and thermostability with no apparent changes in the specific activity, optimum pH, and temperature compared with the wild type. Single site mutations suggested that variant Xyl21-A16 is the cumulative result of three mutated sites, which indicated that K16 and L94 play important roles in enzyme characteristics. Moreover, a comparison of the predicted protein structures of Xyl21 and its variant indicated that additional molecular interactions formed by K16R and K262N might directly improve the rigidity of the protein structure, therefore contributing to the increased thermostability and product tolerance. The variant Xyl21-A16 developed in this study has great application potential in the production of prebiotics, and also provides a useful reference for the future engineering of other xylosidases.

Improving the specific activity and thermostability of psychrophilic xylosidase is important for improving its enzymatic performance and promoting its industrial application. Herein, a psychrophilic xylosidase AX543 exhibited activity in the temperature range between 0 and 35 °C, with optimum activity at 20 °C, which is lower than that of other reported psychrophilic xylosidases. The thermostability, specific activity, and catalytic efficiency of the site-directed variants G110S, Q201R, and L2 were significantly enhanced, without affecting the optimal reaction temperature. Comparative protein structural analysis and molecular dynamics simulation indicated that these improvements might be the result of the increased hydrogen bonds interaction and improved structural rigidity. Furthermore, homologous module substitution with four segments demonstrated that the psychrophilic characteristics of AX543 are the results of the whole protein structure, and the C-terminal segment A4 appears to be more essential in determining psychrophilic characteristics, exhibiting potentiality to produce more psychrophilic xylosidases. This study provides valuable structural information on psychrophilic xylosidases and also offers attractive modification strategies to modify catalytic activity, thermostability, and optimal reaction temperature.

The use of host to secrete several hemicellulase is a cost-effective way for hemicellulose degradation. In this study, the xylose utilization gene xylAB of Escherichia coli BL21 was knocked out, and the xylanase (N20Xyl), β-xylosidase (Xys), and feruloyl esterase (FaeLam) were co-expressed in this strain. By measuring the content of reducing sugars generated by enzymatic hydrolysis of wheat bran in the fermentation supernatant, the order of the three enzymes was screened to obtain the optimal recombinant strain of E. coli BL21/∆xylAB/pDIII-2. Subsequently, fermentation conditions including culture medium, inducer concentration, induction timing, metal ions, and glycine concentration were optimized. Then, different concentrations of wheat bran and xylan were added to the fermentation medium for degradation. The results showed that the extracellular reducing sugars content reached the highest value of 33.70 ± 0.46 g/L when 50 g/L xylan was added. Besides, the scavenging rates of hydroxyl radical by the fermentation supernatant was 81.0 ± 1.41 %, and the total antioxidant capacity reached 2.289 ± 0.55. Furthermore, it showed the growth promotion effect on different lactic acid bacteria. These results provided a basis for constructing E. coli strain to efficiently degrade hemicellulose, and the strain obtained has great potential application to transform hemicellulose into fermentable carbon source. • The xylose utilization gene of E. coli BL21 was knocked out by Red/ET recombination technique. • This strain was used to co-secrete xylosidase Xys, feruloyl esterase FaeLam, and xylanase N20Xyl. • The expression order of Xys, FaeLam, and N20Xyl, and fermentation conditions were optimized. • The recombinant E. coli was used to degrade hemicellulose in synchronous growth and fermentation. • The hydrolysate with high antioxidant activity was used to cultivate Lactic acid bacteria strains. [ABSTRACT FROM AUTHOR]

Xylosidases with tolerance to high concentration of salts, organic solvents, and enzyme hydrolytic products are preferential for industrial application but were rarely reported. In this study, a novel xylosidase XYL21 belong to glycoside hydrolase 39 was characterized with optimal temperature of 45 °C and optimal pH of 5.50. Different to other GH39 xylosidases, XYL21 had excellent tolerance to salts, the activity of which is not inhibited but slightly increased in 0.50–1.50 M NaCl. It is also tolerant to organic solvents, especially retaining 105.18% relative activity even in the presence of 15.00% (v/v) ethanol. Moreover, XYL21 was insensitive to the final lignocellulose hydrolysis products including glucose, xylose, arabinose, mannose and galactose, which retains 111.36% and 53.49% relative activity in 0.30 and 0.90 M xylose, respectively. Further structural modeling analysis indicated that its excellent tolerance may be attributed to its high structural flexibility caused by the high proportion of random coils. Furthermore, XYL21 had a wide substrate specificity to catalyze xylan and xylo-oligosaccharides, and it significantly cooperated with xylanase to improve the hydrolysis efficiency with 1.52-fold. Considering these unique properties, XYL21 is a good candidate for both basic research and various potential industrial applications such as seafood processing and bioethanol production. [ABSTRACT FROM AUTHOR]

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Inhibition of cellulose hydrolysis has been reported extensively, however, there is a paucity of information describing the effect of lignin on xylan hydrolysis by endo-xylanase and β-xylosidase. In this report, the effects of two different lignins on enzymatic hydrolysis of isolated xylan and NaOH-pretreated pennisetum by endo-xylanase and β-xylosidase were assessed. Both acid insoluble lignin (AIL) and enzymatic hydrolysis lignin (EHL) were found to inhibit hydrolysis of endo-xylanase and β-xylosidase, and AIL had a stronger negative effect on enzymatic hydrolysis when compared with that of EHL. Results from inhibitory kinetics experiments showed that the inhibition of AIL and EHL on xylanase did not follow Michaelis–Menten kinetics. The higher adsorption capacity of AIL toward xylanase arose from its higher hydrophobicity and lower absolute zeta potential, and this likely explains the higher inhibitory effect of AIL than that of EHL. The results aid our understanding of the role of lignin in xylan hydrolysis by xylanolytic enzymes. [ABSTRACT FROM AUTHOR]

Highlights • Cellulose, cellobiose and glucose were inhibitors of endoxylanase. • Cellulose, cellobiose and glucose had no inhibitory effect on β-xylosidase. • The order of inhibitory effects was cellobiose > glucose > cellulose. • Cellobiose and glucose were competitive inhibitor of endoxylanase. Abstract The effects of cellobiose and glucose as product inhibitors on the hydrolysis of lignocelluloses by cellulase has been confirmed, but the effect of these species on xylanase is still unknown. In this work, the inhibitory effects of cellulose and its derivatives on xylanase in the hydrolysis of Chinese pennisetum, a potential lignocellulosic biomass for the production of biofuels, was investigated. It was found that cellulose, cellobiose, and glucose all have negative effects on the hydrolytic action of endo-xylanase but not on that of β-xylosidase. Among these inhibitors, cellobiose exhibited the strongest inhibitory effect on xylanase, followed by glucose and cellulose. Cellulose (50 mg/mL), cellobiose (50 mg/mL), and glucose (50 mg/mL) decreased the hydrolysis yields of xylan by 10.4%, 19.7%, and 17.7%, respectively. Kinetic analysis revealed that both cellobiose and glucose competitively inhibited the activity of xylanase. The conclusions of this work help us further understand the inhibitory effects of cellulose derivatives on the hydrolysis of lignocelluloses. [ABSTRACT FROM AUTHOR]

Abstract Endo-xylanase and β-xylosidase are the major enzymes for hemicellulose hydrolysis, which play a significant role in biomass conversion. In our previous work, the white-rot fungi Pleurotus ostreatus HAUCC 162 and Irpex lacteus CD2 were demonstrated to have strong ability in lignocellulose degradation, and the related lignin degradation enzymes were characterized. However, little was known about their hemicellulases. In this work, a novel endo-1, 4-xylanase and a β-xylosidase from Pleurotus ostreatus HAUCC 162 and Irpex lacteus CD2 were heterologously expressed and characterized. The optima of pH and temperature were 5.0 and 55 °C for rXyn162, and 6.5 and 30 °C for rXylCD2. rXyn162 showed high tolerance to metal ions such as Ca2+, Cr3+, Zn2+, Na+, and Al3+. The recombinant rXyn162 and rXylCD2 exhibited synergistic hydrolysis of oat spelts xylan and sodium hydroxide pretreated cornstalk (SHPC), where the degree of synergy (DS) was 2.26 for SHPC hydrolysis. MALDI-TOF-MS and HPLC analysis showed that xylooligosaccharides (XOS) with small degrees of polymerization (DP2-DP4) were the major XOS hydrolyzate during SHPC degradation by rXyn162 and rXylCD2. In addition, rXyn162 and rXylCD2 could efficiently improve the hydrolysis of SHPC by commercial cellulase. The present study suggested the potential application of rXyn162 and rXylCD2 in the field of biomass pretreatment and biofuel production. Graphical abstract Image 1 Highlights • Novel endo-1,4-xylanase and β-xylosidase were heterologously expressed and characterized. • Synergistic hydrolysis of xylan and SHPC by rXyn162 and rXylCD2 was observed. • XOS with small degrees of polymerization were majorly present in SHPC hydrolysate. • rXyn162 and rXylCD2 enhanced SHPC hydrolysis by commercial cellulase. [ABSTRACT FROM AUTHOR]

Synergism between enzymes is of crucial importance in cell metabolism. This synergism occurs often through a spatial organisation favouring proximity and substrate channelling. In this context, we developed a strategy for evaluating the impact of the geometry between two enzymes involved in nature in the recycling of the carbon derived from plant cell wall polymers. By using an innovative covalent association process using two protein fragments, Jo and In, we produced two bi-modular chimeric complexes connecting a xylanase and a xylosidase, involved in the deconstruction of xylose-based plant cell wall polymer. We first show that the intrinsic activity of the individual enzymes was preserved. Small Angle X-rays Scattering (SAXS) analysis of the complexes highlighted two different spatial organisations in solution, affecting both the distance between the enzymes (53 Å and 28 Å) and the distance between the catalytic pockets (94 Å and 75 Å). Reducing sugar and HPAEC-PAD analysis revealed different behaviour regarding the hydrolysis of Beechwood xylan. After 24 h of hydrolysis, one complex was able to release a higher amount of reducing sugar compare to the free enzymes (i.e., 15,640 and 14,549 µM of equivalent xylose, respectively). However, more interestingly, the two complexes were able to release variable percentages of xylooligosaccharides compared to the free enzymes. The structure of the complexes revealed some putative steric hindrance, which impacted both enzymatic efficiency and the product profile. This report shows that controlling the spatial geometry between two enzymes would help to better investigate synergism effect within complex multi-enzymatic machinery and control the final product.

The Xylo-1 xylosidase, which belongs to the GH43 family, exhibits a high salt tolerance. The present study demonstrated that the catalytic activity of Xylo-1 increased by 195% in the presence of 5 M NaCl. Additionally, the half-life of Xylo-1 increased 25.9-fold in the presence of 1 M NaCl. Through comprehensive analysis including circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations, we elucidated that the presence of Na + ions increased the contact frequency between the surface acidic amino acids and the surrounding water molecules. This resulted in the stabilization of the surrounding hydration layer of Xylo-1. Additionally, Na + ions also stabilized the substrate-binding conformation and the fluctuation of water molecules within the active site, which enhanced the catalytic activity of Xylo-1 by increasing the nucleophilic attack by the water molecules. Ultimately, the optimal reaction conditions for the production of xylose by synergistic catalysis with Xylo-1 and xylanase were determined. The results demonstrated that the conversion yield of the method was high for various sources of xylan, indicating the method could have potential industrial applications. This study explored the structure-activity relationship of catalysis in Xylo-1 under high-salt conditions, provides novel insights into the mechanism of halophilic enzymes, and serves as a reference for the industrial application of Xylo-1.

Xylanase and β -xylosidase are the key enzymes for hemicellulose hydrolysis. To further improve hydrolysis efficacy, high temperature hydrolysis with thermostable hemicellulases showed promise. In this study, thermostable xylanase (Xyn) and β -xylosidase (XynB) genes from Pseudothermotoga thermarum were cloned and secretory expressed in Bacillu subtilis . Compared with Escherichia coli expression host, B. subtilis resulted in a 1.5 time increase of enzymatic activity for both recombinant enzymes. The optimal temperature and pH were 95°C and 6.5 for Xyn, and 95°C and 6.0 for XynB. Thermostability of both recombinant enzymes was observed between the temperature range of 75-85°C. Molecular docking analysis through AutoDock showed the involvement of Glu525, Asn526, Trp774 and Arg784 in Xyn-ligand interaction, and Val237, Lys238, Val761 and Asn76 in XynB-ligand interaction, respectively. The recombinant Xyn and XynB exhibited synergistic hydrolysis of beechwood xylan and pretreated lignocellulose, where Xyn and XynB pre-hydrolysis achieved a better improvement of pretreated lignocellulose hydrolysis by commercial cellulase. The observed stability of the enzymes at high temperature and the synergistic effect on lignocellulosic substrates suggested possible application of these enzymes in the field of saccharification process., Competing Interests: Author HQ was employed by Little Swan Electric Co., Ltd., Midea Group. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Qin, You and Long.)

Two GH43 β-xylosidases, RS223-BX from a rice straw metagenomic library, and BoXA from Bacteroides ovatus , that share similar amino acid sequences (81% identical) and 19 of 20 active-site residues, were compared by using site-directed mutagenesis of Asp and His residues implicated in metal binding. Thus, RS223-BX is strongly activated by divalent-metal cations and the previously published X-ray structure of this enzyme shows that a Ca 2+ cation is chelated by an active-site Asp carboxyl group and an active-site His. Mutation to Ala causes 90% loss of activity for the Asp mutant and 98% loss of activity for the His mutant, indicating their importance to catalysis. For the other enzyme (BoXA), mutation to Ala causes 20% loss of activity for the His mutant and 40% gain of activity for the Asp mutant, indicating the lack of importance for activity of the native residues and the lack of metal-dependency, given that the Asp residue occupies the active site to secure the metal cation in known metal ion dependent GH43 xylosidases. The high activity of the BoXA mutants compared to that of the analogous RS223-BX mutants further undermines the possibility that BoXA maintains a tightly bound metal cofactor resistant to EDTA extraction. The results strengthen our conclusion that the very similar proteins differ in one being metal ion dependent and one not. [ABSTRACT FROM AUTHOR]

The gene of Pae1263 (2,196 bp, 732 aa) was found from the full-length sequence analysis of bacterium Paenibacillus terrae HPL-003 isolated from soil on Gara Mountain in Korea (CP003107, our previous study). Among the 20 open reading frames (ORFs) related with the xylose substrate, only the recombinant enzyme of ORF Pae1263 showed a 1,4-beta-D-xylosidase activity when all of the ORFs were transformed into E. coli. This gene is considered to be a new 1,4-beta-D-xylosidase because it has up to 93% similarity with other genes of ZP_10240221.1 from Lactococcus raffinolactis 4877 and ZP_11237858.1 from Paenibacillus peoriae in the GenBank blast search. The enzyme activity was confirmed by HPLC in which xylose was produced from xylobiose as a substrate by this recombinant enzyme. Mass production of the recombinant enzyme was done with the construction of the pET22(+)- Pae1263-6H expression vector system from E. coli. This new 1,4-beta-D-xylosidase was highly active at 50°C in a pH range between 6.0 and 8.0 and had thermo-stability for at least 24 h at 50°C and a Km and Vmax of 6.42 mg/mL and 75.76 U/mg on a xylobiose substrate, respectively. [ABSTRACT FROM AUTHOR]

Endo-1, 4-β-xylanase is an enzyme that depolymerises xylan, a major component of lignocellulose. The economic utilization of lignocellulosic biomass as a feedstock for the production of fuel and chemicals would represent a paradigm shift in industrial carbon utilization, allowing sustainable resources to replace the non-renewable energy resources like petroleum. However, lignocellulose is a highly recalcitrant material that is extremely difficult to depolymerize. Many microorganisms possess repertoires of enzymes which act synergistically to decompose various components of lignocellulosic biomass into simple sugars. Biomass-utilizing organisms are widely distributed within different species of archaea, bacteria, fungi, protists, plants, and animals. These organisms possess numerous lignocellulolytic enzymes that can hydrolyze cellulose, hemicelluloses, or lignin. The present paper gives a detailed account of strategies for production, purification and application of microbial xylanases. Conversion of lignocellulose into fermentable sugars and subsequent conversion into bioethanol has been discussed in the present paper. [ABSTRACT FROM AUTHOR]

Xylooligosaccharide-assimilating Corynebacterium glutamicum strains were constructed using metabolic engineering and cell surface display techniques. First, C. glutamicum was metabolically engineered to create lysine-producing strains. Beta-xylosidase BSU17580 derived from Bacillus subtilis was then expressed on the C. glutamicum cell surface using PorH anchor protein, and enzymes involved in the xylose assimilation pathway were also expressed. Metabolic engineering had no effect on the activity of beta-xylosidase. The engineered strains efficiently consumed xylooligosaccharides and produced 12.4 mM of lysine from 11.9 g/L of xylooligosaccharides as the carbon source. Finally, co-expression of lysine decarboxylase enabled production of 11.6 mM of 1,5-diaminopentane (cadaverine) from 13 g/L of consumed xylooligosaccharides. [ABSTRACT FROM AUTHOR]

A fungal endoxylanase belonging to the glycoside hydrolase gene family 11 (GH11) was obtained from the ascomycete Talaromyces amestolkiae. The enzyme was purified, characterized and used to produce a mixture of xylooligosaccharides (XOS) from birchwood xylan. A notable yield of neutral XOS was obtained (28.8%) upon enzyme treatment and the mixture contained a negligible amount of xylose, having xylobiose, xylotriose and xylotetraose as its main components. The prebiotic potential of this mixture was demonstrated upon analyzing the variations in microorganisms’ composition and organic acids profile in breast-fed child faeces fermentations. The strong production of acetic and lactic acid, the decrease of potentially pathogenic bacteria and the increase of bifidobacteria, and possible beneficial commensals, confirmed the prebiotic value of these xylooligosaccharides. [ABSTRACT FROM AUTHOR]

Corn cobs (CCs) are abundant xylan-rich agricultural wastes. Here, we compared CCs XOS yields obtained via two different pretreatment routs, alkali and hydrothermal, using a set of recombinant endo - and exo -acting enzymes from GH10 and GH11 families, which have different restrictions for xylan substitutions. Furthermore, impacts of the pretreatments on chemical composition and physical structure of the CCs samples were evaluated. We demonstrated that alkali pretreatment route rendered 59 mg of XOS per gram of initial biomass, while an overall XOS yield of 115 mg/g was achieved via hydrothermal pretreatment using a combination of GH10 and GH11 enzymes. These results hold a promise of ecologically sustainable enzymatic valorization of CCs via "green" and sustainable XOS production. [Display omitted] [ABSTRACT FROM AUTHOR]

Key message: The results obtained indicate that a β-xylosidase gene may act as good indicator of chilling tolerance and provide new insights into the complex issue of peach fruit woolliness. Abstract: The storage of peaches at low temperatures for prolonged periods can induce a form of chilling injury (CI) called woolliness, characterized by a lack of juiciness and a mealy texture. As this disorder has been associated with abnormal cell wall dismantling, the levels of 12 transcripts encoding proteins involved in cell wall metabolism were analysed in cultivars with contrasting susceptibility to this disorder selected from five melting flesh peach cultivars. The resistant ('Springlady') and susceptible ('Flordaking') cultivars displayed differences in the level of expression of some of the selected genes during fruit softening and in woolly versus non-woolly fruits. From these genes, the level of expression of PpXyl, which encodes for a putative β-xylosidase, was the one that presented the highest correlation (negative) with the susceptibility to woolliness. PpXyl expression was also analysed in a cultivar ('Rojo 2') with intermediate susceptibility to woolliness, reinforcing the conclusion about the correlation of PpXyl expression to the presence of woolliness symptom. Moreover, the level of expression of PpXyl correlated to protein level detected by Western blot. Analyses of the promoter region of the PpXyl gene (1637 bp) isolated from the three cultivars showed no differences suggesting that cis-elements from other regions of the genome and/or trans elements could be responsible of the differential PpXyl expression patterns. Overall, the results obtained indicate that PpXyl may act as a good indicator of woolliness tolerance and that the regulation of expression of this gene in different cultivars does not depend on sequences upstream the coding sequence. [ABSTRACT FROM AUTHOR]

In this work, we present the first XOS degrading glycoside hydrolase from Weissella, WXyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species-pair Weissella cibaria/W.confusa, and expressed in Escherichia coli. The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold β-propeller catalytic domain and a C-terminal β-sandwich domain of unknown function.WXyn43 hydrolyzed short (1-4)-β-D-xylooligosaccharides, with similar kcat/KM for xylobiose (X2) and xylotriose (X3) and clearly lower efficiency in xylotetraose (X4) conversion. WXyn43 displays the highest reported kcat for conversion of X3 (900 s-1 at 37°C) and X4 (770 s-1), and kcat for hydrolysis of X2 (907 s-1) is comparable with or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer-monomer hydrogen bonds, while the bonds creating dimer-dimer interactions only involved residues from the N-terminal domain. Docking of X2 and X3 in the active site shows interactions corresponding to subsites -1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X3 may be present. [ABSTRACT FROM AUTHOR]

Background: Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomass degradation are based on the use of single substrates. Moreover, highly diverse environments are used as metagenomic sources. However, such methods suffer from low hit rates of positive clones and hence the discovery of novel enzymatic activities from metagenomes has been hampered. Results: Here, we constructed fosmid libraries from two wheat straw-degrading microbial consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw). Approximately 22,000 clones from each library were screened for (hemi)cellulose-degrading enzymes using a multi-chromogenic substrate approach. The screens yielded 71 positive clones for both libraries, giving hit rates of 1:440 and 1:1,047 for RWS and TWS, respectively. Seven clones (NT2-2, T5-5, NT18-17, T4-1, 10BT, NT18-21 and T17-2) were selected for sequence analyses. Their inserts revealed the presence of 18 genes encoding enzymes belonging to twelve different glycosyl hydrolase families (GH2, GH3, GH13, GH17, GH20, GH27, GH32, GH39, GH53, GH58, GH65 and GH109). These encompassed several carbohydrate-active gene clusters traceable mainly to Klebsiella related species. Detailed functional analyses showed that clone NT2-2 (containing a beta-galactosidase of ~116 kDa) had highest enzymatic activity at 55 °C and pH 9.0. Additionally, clone T5-5 (containing a beta-xylosidase of ~86 kDa) showed > 90 % of enzymatic activity at 55 °C and pH 10.0. Conclusions: This study employed a high-throughput method for rapid screening of fosmid metagenomic libraries for (hemi)cellulose-degrading enzymes. The approach, consisting of screens on multi-substrates coupled to further analyses, revealed high hit rates, as compared with recent other studies. Two clones, 10BT and T4-1, required the presence of multiple substrates for detectable activity, indicating a new avenue in library activity screening. Finally, clones NT2-2, T5-5 and NT18-17 were found to encode putative novel thermo-alkaline enzymes, which could represent a starting point for further biotechnological applications. [ABSTRACT FROM AUTHOR]

In this work, the roles of hemicellulases, endoxylanase and β-xylosidase, in improving the hydrolysis of corn stover pretreated by aqueous ammonia (CS–AA) and dilute acid (CS–DA) were evaluated. Synergistic actions of endoxylanase and β-xylosidase were observed in the release of xylose in the hydrolysis of both isolated xylan and xylan in pretreated corn stover. Endoxylanase significantly reduced the negative effect of xylan on the action of cellulases, especially on cellobiohydrolase I. The addition of β-xylosidase from Selenomonas ruminantium increased the xylose yields from 9.6% and 13.0% to 31.7% and 47.6% in the hydrolysis of CS–AA by cellulases and xylanase at 40 °C and 50 °C, respectively. Furthermore, the addition of thermostable β-xylosidase from Entamoeba coli increased glucose yields from 40.3% and 20.7% to 44.0% and 26.6% in the hydrolysis of CS–AA and CS–DA by cellulases and xylanase at 50 °C, respectively. β-xylosidase significantly reduced xylo-oligosaccharides inhibition on cellobiohydrolase I by converting most of xylo-oligosaccharides (93.6%) to the less inhibitory xylose, showing the importance and potential benefits of β-xylosidase in efficient and complete hydrolysis of lignocelluloses. [ABSTRACT FROM AUTHOR]

Streptomyces griseorubens JSD-1 is an isolate that can utilize lignocellulose (straw) as its sole carbon source for growth, and these lignocellulolytic genes involved in this biotransformation are expected to be crucial. However, little is known about the genetic basis related to this process. To further investigate the lignocellulose-degrading mechanisms, genome sequencing was carried out using MiSeq platform. After obtaining its draft genome, the key lignocellulolytic genes such as multicopper oxidase, exo-1, 4-β-glucanase, endo-1,4-β-glucanase, and β-xylosidase were identified and characterized. Multiple sequence alignments were performed to find out the identities of these analyzed proteins to those of their similar species. Signal peptide cleavage sites were predicted by SignalP 4.1 to reflect the cellular localization of their mature proteins. Besides, predicted 3D structures of these proteins were modeled by Phyre2, which showed to be highly identical to the templates in the PDB database. Finally, quantitative real-time PCR revealed that expression levels of all analyzed enzymes were significantly and generally up-regulated during the whole cultivation, indicating that they all contributed to the biodegradation. [ABSTRACT FROM AUTHOR]

Xylan is an abundant plant cell wall polysaccharide and its reduction to xylose units for subsequent biotechnological applications requires a combination of distinct hemicellulases and auxiliary enzymes, mainly endo-xylanases and ß-xylosidases. In the present work, a bifunctional enzyme consisting of a GH11 endo-1,4-β-xylanase fused to a GH43 β-xylosidase, both from Bacillus subtilis , was designed taking into account the quaternary arrangement and accessibility to the substrate. The parental enzymes and the resulting chimera were successfully expressed in Escherichia coli , purified and characterized. Interestingly, the substrate cleavage rate was altered by the molecular fusion improving at least 3-fold the xylose production using specific substrates as beechwood xylan and hemicelluloses from pretreated biomass. Moreover, the chimeric enzyme showed higher thermotolerance with a positive shift of the optimum temperature from 35 to 50 °C for xylosidase activity. This improvement in the thermal stability was also observed by circular dichroism unfolding studies, which seems to be related to a gain of stability of the β-xylosidase domain. These results demonstrate the superior functional and stability properties of the chimeric enzyme in comparison to individual parental domains, suggesting the molecular fusion as a promising strategy for enhancing enzyme cocktails aiming at lignocellulose hydrolysis. [ABSTRACT FROM AUTHOR]

Geobacillus stearothermophilus T6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of β-xylosidases. One of these β-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombic P212121 space group, with average unit-cell parameters a = 97.7, b = 119.1, c = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Å resolution), the E335G catalytic mutant (2.95 Å resolution), a potential mercury derivative (2.15 Å resolution) and a selenomethionine derivative (3.90 Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein. [ABSTRACT FROM AUTHOR]

Abstract: It has been considered that more efficient uptake of xylose could promote increased xylose metabolic capacity of several microorganisms. In this study, an assay to screen xylose transporters was established in the Saccharomyces cerevisiae strain, which expresses the xylosidase gene of Bacillus pumilus intracellularly. The absorbed xylose analog p-nitrophenyl-β-d-xylopyranoside (pNPX) rapidly hydrolyzed to p-nitrophenol (pNP), which displayed a yellow tint when exposed to xylosidase in vivo. The xylose transporter activities of the strain were computed using the pNP production rate, which was detected extracellularly. This method could be used for both high-throughput screening and smaller scale investigations. AraEp, which is a pentose transporter of Corynebacterium glutamicum, was expressed in S. cerevisiae and exhibited better transport capacity than the endogenous transporters Hxt7p and Gal2p. Moreover, a mutant of AraEp with 103% greater transport capacity was screened out, and the computer simulation suggested that transmembrane domain 5 was an important factor for the transport capacity of AraEp in S. cerevisiae. [Copyright &y& Elsevier]

Six strains isolated from fermented food were identified as Weissella species by 16 S r DNA sequencing, clustering with the species pair W. confusa/ W. cibaria. The strains were analysed for growth on glucose, xylose and xylooligosaccharides ( XOS). All strains were xylose positive using the API CHL 50 test. Growth on XOS was observed for strains 85, 92, 145 and AV1, firstly by optical density measurements in microtitre plates and secondly in batch cultures also confirming concomitant decrease in p H. Analysis of XOS before and after growth established consumption in the DP2- DP5 range in the four XOS-fermenting strains. XOS were consumed simultaneously with glucose, while xylose was consumed after glucose depletion. Cell-associated β-xylosidase activity was detected in the XOS-fermenting strains. Analysis of genomic data suggests this activity to be linked with genes encoding glycoside hydrolases from family 3, 8 or 43. No endo-β-xylanase activity was detectable. Major end products were lactate and acetate. A higher ratio of acetic acid to lactic acid was obtained during growth on XOS compared with growth on glucose. This is the first report on utilization of XOS in Weissella, indicating an increased probiotic potential for XOS-utilizing strains from the species pair W. confusa/ W. cibaria, but also showing that XOS utilization is strain dependent for these species. [ABSTRACT FROM AUTHOR]

A bifunctional xylosidase/arabinofuranosidase gene (PcXyl) was cloned from the cDNA library of Phanerochaete chrysosporium and further expressed in Pichia pastoris. Enzymatic assay indicated that P. pastoris produced rPcXyl at a level of 26,141 U l−1. The xylosidase and arabinofuranosidase activities of rPcXyl were maximized, respectively, at pHs of 5.0 and 5.5 and temperatures of 45°C and 50°C. SDS-PAGE revealed a single band of purified rPcXyl of 83 kDa. Cu2+ and Zn2+ completely inhibited the enzyme activity of rPcXyl. The enzyme activity of rPcXyl was increased 151%, 126% and 123%, respectively, in the presence of glucose, xylose and arabinose at concentrations of 5 mM. rPcXyl hydrolyzed xylobiose to xylose and xylotriose to xylose and xylobiose, indicating rPcXyl acts as an exo-type enzyme. Additionally, rPcXyl enhanced xylose release from xylan substrates in synergy with rPcXynC. [ABSTRACT FROM AUTHOR]