xylobiose and xylotriose

Propionic acid (PA) hydrolysis offers a potential pathway for industrial xylooligosaccharide (XOS) production owing to efficiency and simplicity of the process. However, the cost of XOS production needs to be reduced as PA is expensive. This work proposed a strategy of mixed acids hydrolysis, replacing 20% of PA with formic acid (FA), and combined with xylanase hydrolysis to reduce production costs and increase the production of XOS from corncob. The hydrolysis of corncob using mixed FA and PA in a mass ratio of 2:8 produced 61.8% XOS. Xylanase hydrolysis of corncob residue improved XOS yield to 73.1%. Among them, the X2 + X3 yield was as high as 50.6%. Economic evaluation showed that the combined process reduced the XOS production cost by 10.8% compared to PA hydrolysis. The strategy of using FA instead of 20% PA for hydrolysis and enzymatic hydrolysis, with high XOS and monosaccharide yields from corncob, has potential industrial promise.

Topics: Disaccharides; Hydrolysis; Zea mays

Xylooligosaccharides having various degrees of polymerization such as xylobiose, xylotriose, and xylotetraose positively affect human health by interacting with gut proteins. The present study aimed to identify proteins present in gut microflora, such as xylosidase, xylulokinase, etc., with the help of retrieved whole-genome annotations and find out the mechanistic interactions of those with the above substrates. The 3D structures of proteins, namely Endo-1,4-beta-xylanase B (XynB) from Lactobacillus brevis and beta-D-xylosidase (Xyl3) from Bifidobacterium adolescentis, were computationally predicted and validated with the help of various bioinformatics tools. Molecular docking studies identified the effectual binding of these proteins to the xylooligosaccharides, and the stabilities of the best-docked complexes were analyzed by molecular dynamic simulation. The present study demonstrated that XynB and Xyl3 showed better effectual binding toward Xylobiose with the binding energies of - 5.96 kcal/mol and - 4.2 kcal/mol, respectively. The interactions were stabilized by several hydrogen bonding having desolvation energy (- 6.59 and - 7.91). The conformational stabilities of the docked complexes were observed in the four selected complexes of XynB-xylotriose, XynB-xylotetraose, Xyl3-xylobiose, and Xyn3-xylotriose by MD simulations. This study showed that the interactions of these four complexes are stable, which means they have complex metabolic activities among each other. Extending these studies of understanding, the interaction between specific probiotics enzymes and their ligands can explore the detailed design of synbiotics in the future.

Topics: Bacterial Proteins; Bifidobacterium adolescentis; Computational Biology; Disaccharides; Endo-1,4-beta Xylanases; Genome, Bacterial; Glucuronates; Humans; Levilactobacillus brevis; Molecular Docking Simulation; Molecular Dynamics Simulation; Oligosaccharides; Probiotics; Trisaccharides; Xylosidases

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.

Topics: Animals; Bacteroidetes; Bifidobacterium adolescentis; Disaccharides; Endo-1,4-beta Xylanases; Fermentation; Glucuronates; Humans; Hydrolysis; Oligosaccharides; Prebiotics; Trisaccharides; Xylans; Xylose; Zea mays

The prebiotic properties of xylooligosaccharides (XOS) and arabino-xylooligosaccharides (AXOS) produced from rice husk (RH) using microwave treatment combined with enzymatic hydrolysis were evaluated. The RH was subjected to microwave pretreatment at 140, 160 and 180 °C for 5, 10 and 15 min to obtain crude arabinoxylan (AX). Increasing microwave pretreatment time increased sugar content. Crude AX was extracted with 2% (w/v) sodium hydroxide at 25 °C for 24 h and used as a substrate for XOS production by commercial xylanases. Results showed that oligosaccharides produced by Pentopan Mono BG and Ultraflo Max provided xylobiose and xylotriose as the main products. AXOS was also present in the oligosaccharides that promoted growth of Lactobacillus spp. and resisted degradation by over 70% after exposure to simulated human digestion.

Topics: Alkalies; Disaccharides; Endo-1,4-beta Xylanases; Glucuronates; Hydrolysis; Microwaves; Oligosaccharides; Oryza; Prebiotics; Seeds; Trisaccharides; Xylans

A promising approach for production of value-added xylooligosaccharides (XOS) from poplar was developed by combining hydrothermal pretreatment and endo-xylanase post-hydrolysis. Results showed that the 35.4% XOS (DP 2-6) and 17.6% low DP xylans (DP > 6) were obtained at the identified optimal condition (170 °C, 50 min) for hydrothermal pretreatment. Structural features of low DP xylans generated during the hydrothermal pretreatment were examined, revealing that low DP xylans are mainly comprised of 4-O-methylglucuronic xylan and are involved in lignin carbohydrate complexes. Moreover, higher pretreatment intensity promoted the cleavage of side-chain substituents including arabinose and glucuronic acid groups. The subsequent endo-xylanase hydrolysis of the pretreatment liquor hydrolyzed low DP xylans, contributing to a significant improvement in xylobiose and xylotriose proportions. This combined strategy resulted in a XOS with conversion yield of 44.6% containing 78.7% xylobiose and xylotriose starting from the initial xylan in raw poplar.

Topics: Disaccharides; Endo-1,4-beta Xylanases; Glucuronates; Hydrolysis; Oligosaccharides; Trisaccharides; Xylans

Here, we proposed an effective strategy to enhance a novel endoxylanase (Taxy11) activity and elucidated an efficient catalysis mechanism to produce xylooligosaccharides (XOSs). Codon optimization and recruitment of natural propeptide in Pichia pastoris resulted in achievement of Taxy11 activity to 1405.65 ± 51.24 U/mL. Analysis of action mode reveals that Taxy11 requires at least three xylose (xylotriose) residues for hydrolysis to yield xylobiose. Results of site-directed mutagenesis indicate that residues Glu

Topics: Cloning, Molecular; Disaccharides; Endo-1,4-beta Xylanases; Hydrolysis; Kinetics; Pichia; Substrate Specificity; Trichoderma; Trisaccharides; Xylan Endo-1,3-beta-Xylosidase; Xylans; Xylose

A one-step method to immobilize xylanase onto cellulosic material by fusion of expansin from Bacillus subtilis to xylanase LC9 without the requirement of prior purification of enzyme has been developed. Fusion enzyme EXLX-R2-XYN was specifically adsorbed onto corncob residue with high loading capacity due to bio-affinity adsorption of expansin onto cellulose. The immobilization yield was close to 100%, with a recovered activity of 82.4%. The immobilized EXLX-R2-XYN retained 45.3% of its activity after incubation at 70 °C for 3 h, whereas only 16.3% of the activity was left in free form under the same conditions. The conversion yield of XOS by using immobilized EXLX-R2-XYN reached up to 515 mg/g xylan from 2% corncob extracted xylan, which was higher than that of the free enzyme. The hydrolysis products were mainly xylobiose (57.5%) and xylotriose (38.4%), without undesirable xylose production. After five cycles of hydrolysis, more than 70% of conversion was obtained.

Topics: Bacillus subtilis; Bacterial Proteins; Chromatography, Affinity; Disaccharides; Endo-1,4-beta Xylanases; Enzymes, Immobilized; Glucuronates; Hydrogen-Ion Concentration; Hydrolysis; Oligosaccharides; Plant Proteins; Recombinant Fusion Proteins; Recycling; Temperature; Trisaccharides

Xylanases are enzymes that act in the depolymerization of xylan and that can be used in the food industry, the paper industry, and for bioenergy, among other uses. In this context, particular emphasis is devoted to xylooligosaccharides (XOS) that act as prebiotics, which, under the action of probiotic microorganisms, are capable of positively modifying the intestinal microbiota. In this sense, searching for microbial xylanases stands out as a sustainable strategy for the production of prebiotics. To date, there have been no reports in the literature regarding the purification of native xylanase from Myceliophthora heterothallica F.2.1.4. In this study, a xylanase from this fungus was purified and characterized. The xylanase, with 27 kDa, showed maximum activity at pH 4.5 and 65-70 °C. It maintained more than 80% of its residual activity when exposed to (i) temperatures between 30 and 60 °C for 1 h and (ii) pH 5-10 for 24 h at 4 and 25 °C. These high tolerances to different pH and different temperatures are important properties that add value to this enzyme. The hydrolysates of this enzyme on beechwood xylan, analyzed by HPAE-PAD, were mostly xylobiose (X2) and xylotriose (X3). Hydrolysates were also quantified, being retrieved from 234.2 mg xylooligosaccharides/g of hydrolyzed xylan for 12 h. According to the products obtained from the xylan hydrolysis and its tolerance properties of the enzyme, it has demonstrated potential for application production of xylooligosaccharides for use as prebiotics.

Topics: Ascomycota; Disaccharides; Endo-1,4-beta Xylanases; Enzyme Stability; Glucuronates; Oligosaccharides; Probiotics; Temperature; Trisaccharides

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-L-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-L-arabinofuranoside with k

Topics: Arabinose; Corynebacterium; Corynebacterium glutamicum; Disaccharides; Endo-1,4-beta Xylanases; Escherichia coli; Glucuronates; Glycoside Hydrolases; Hydrolysis; Oligosaccharides; Recombinant Proteins; Trisaccharides; Xylans; Xylose

A bio-refinery process of wheat straw pulping solid residue (waste wheat straw, WWS) was established by combining prewashing and liquid hot water pretreatment (LHWP). The results showed that employing a prewashing step prior to the LHWP remarkably improved enzymatic glucose yields from 39.7% to 76.6%. Moreover, after 96h simultaneous saccharification and fermentation (SSF), identical ethanol yields of 0.41g/g-cellulose were obtained despite varied solid loadings (5-30%). Beyond ethanol, enzymatic post-hydrolysis of the prehydrolyzate effectively increased xylobiose and xylotriose yields from 15mg/g-WWS and 14mg/g-WWS to 53mg/g-WWS and 20mg/g-WWS, respectively. For mass balance, about 10.9tons raw WWS will be consumed to produce 1ton ethanol, in addition to producing 614.8kg xylooligosaccharides (XOS) containing 334.3kg xylobiose and 124.8kg xylotriose. The results demonstrated that the integrated process for the WWS bio-refinery is promising, based on value-adding co-production in addition to robust ethanol yields.

Topics: Biofuels; Disaccharides; Ethanol; Fermentation; Glucuronates; Hydrolysis; Oligosaccharides; Trisaccharides; Triticum

Xylooligosaccharides as emerging prebiotics are able to promote the growth of probiotic bacteria. In the present study, four neutral, thermostable xylanases (MtXyn11A, MtXyn11At, MtXyn11B, and MtXyn11C) from compost fungus Mycothermus thermophilus CGMCC3.18119 were overexpressed in Pichia pastoris GS115 and used to produce xylooligosaccharides from beechwood xylan. The enzymes showed similar enzymatic properties (maximal activities at pH 6.0-6.5 and 65 °C) but varied in catalytic efficiency and cleaving actions. MtXyn11A, MtXyn11At, and MtXyn11C mainly produced xylobiose (59-62%), xylose (16-20%), and xylotriose (16-19%), while MtXyn11B released xylobiose (51%), xylotriose (32%), and xylose (12%) as the main products. When using the xylan hydrolysates of different xylanases as the carbon source, four probiotic Lactobacillus strains Lactobacillus brevis 1.2028, Lactobacillus rhamnosus GG, Lactobacillus casei BL23, and Lactobacillus plantarum WCSF1 were confirmed to use the xylooligosaccharides efficiently (83.8-98.2%), with L. brevis 1.2028 as the greatest.

Topics: Ascomycota; Biotechnology; Disaccharides; Endo-1,4-beta Xylanases; Enzyme Stability; Fungal Proteins; Glucuronates; Hydrolysis; Lactobacillus; Levilactobacillus brevis; Oligosaccharides; Pichia; Prebiotics; Probiotics; Recombinant Proteins; Trisaccharides; Wood; Xylans

In this study, a co-production of two high value-added products, glucose and xylooligosaccharides (XOS), was investigated by utilizing sugarcane bagasse (SB) within a multi-product bio-refinery framework optimized by Box-Behnken design-based response surface methodology. The developed process resulted in a maximum cellulose conversion of xylan-removed SB, 98.69±1.30%, and a maximum extracted SB xylan conversion into XOS (xylobiose and xylotriose) of 57.36±0.79% that was the highest SB xylan conversion reported in the literature, employing cellulase from Penicillium oxalicum EU2106 and recombinant endo-β-1,4-xylanase in Pichia pastoris. Consequently, a mass balance analysis showed that the maximum yields of glucose and XOS were 34.43±0.32g and 5.96±0.09 g per 100 g raw SB. Overall, this described process may be a preferred option for the comprehensive utilization of SB.

Topics: Biotechnology; Cellulase; Cellulose; Disaccharides; Endo-1,4-beta Xylanases; Glucose; Glucuronates; Hydrolysis; Oligosaccharides; Penicillium; Pichia; Saccharum; Trisaccharides

The structure of xylan, which has a 1,4-linked β-xylose backbone with various substituents, is much more heterogeneous and complex than that of cellulose. Because of this, complete degradation of xylan needs a large number of enzymes that includes GH10, GH11, and GH3 family xylanases together with auxiliary enzymes. Fluorescence-assisted carbohydrate electrophoresis (FACE) is able to accurately differentiate unsubstituted and substituted xylooligosaccharides (XOS) in the heterogeneous products generated by different xylanases and allows changes in concentrations of specific XOS to be analyzed quantitatively. Based on a quantitative analysis of XOS profiles over time using FACE, we have demonstrated that GH10 and GH11 family xylanases immediately degrade xylan into sizeable XOS, which are converted into smaller XOS in a much lower speed. The shortest substituted XOS produced by hydrolysis of the substituted xylan backbone by GH10 and GH11 family xylanases were MeGlcA(2) Xyl3 and MeGlcA(2) Xyl4 , respectively. The unsubstituted xylan backbone was degraded into xylose, xylobiose, and xylotriose by both GH10 and GH11 family xylanases; the product profiles are not family-specific but, instead, depend on different subsite binding affinities in the active sites of individual enzymes. Synergystic action between xylanases and β-xylosidase degraded MeGlcA(2) Xyl4 into xylose and MeGlcA(2) Xyl3 but further degradation of MeGlcA(2) Xyl3 required additional enzymes. Synergy between xylanases and β-xylosidase was also found to significantly accelerate the conversion of XOS into xylose.

Topics: Carbohydrates; Disaccharides; Electrophoresis; Endo-1,4-beta Xylanases; Fluorescence; Glucuronates; Oligosaccharides; Trisaccharides; Xylose; Xylosidases

Xylobiose (X2), which is currently available from xylooligosaccharides (XOS), has been reported to have outstanding prebiotic function and to be highly suitable for application in food industries. This has sparked an interest in the economical production of X2 of high purity (> 99%) in food and prebiotic industries. To address such issue, we developed a highly-efficient chromatographic method for the recovery of X2 from XOS with high purity and high recovery. As a first step for this work, an eligible adsorbent for a large-scale separation between X2 and other XOS components was selected. For the selected adsorbent, a single-column experiment was carried out to determine the intrinsic parameters of all the XOS components, which were then used in the optimal design of the continuous X2-recovery process based on a simulated moving bed (SMB) chromatographic method. Finally, the performance of the designed X2-recovery SMB process was verified by the relevant SMB experiments, which confirmed that the developed process in this study could recover X2 from XOS with the purity of 99.5% and the recovery of 92.3% on a continuous-separation mode. The results of this study will be useful in enabling the economical production of high-purity X2 on a large scale.

Topics: Adsorption; Arabinose; Chromatography, High Pressure Liquid; Disaccharides; Glucose; Glucuronates; Oligosaccharides; Trisaccharides; Xylose

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49 %. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0-4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2 % (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.

Topics: Biotechnology; Chelating Agents; Disaccharides; Endo-1,4-beta Xylanases; Enzyme Stability; Enzymes, Immobilized; Fermentation; Fungal Proteins; Glucuronates; Hydrogen-Ion Concentration; Hydrolysis; Nickel; Oligosaccharides; Penicillium; Pichia; Polymers; Recombinant Proteins; Temperature; Trisaccharides; Xylans; Zea mays

Pigeon pea (Cajanus cajan) is a perennial plant widely cultivated in tropical and subtropical regions of many countries. The present studies aimed to produce xylooligosaccharides (XOS) from pigeon pea stalks in order to do value addition. The chemical analysis of stalks revealed 18.33 ± 1.40 % hemicelluloses in addition to cellulose, protein, and lignin. Sodium hydroxide coupled with steam application enabled almost 96 % recovery of original xylan, present in the pigeon pea stalks. Enzymatic hydrolysis of xylan led to production of XOS namely, xylobiose and xylotriose. Response surface model indicated a maximum yield of xylobiose (0.502 mg/ml) under the hydrolysis conditions of pH 4.91, temperature at 48.11 °C, enzyme dose at 11.01 U, and incubation time at 15.65 h. The ideal conditions for higher xylotriose yield (0.204 mg/ml) were pH 5.44, temperature at 39.29 °C, enzyme dose at 3.23 U, and incubation time at 15.26 h. The present investigation was successful in assessing the prospect of using pigeon pea stalks as a raw material for xylan extraction vis-à-vis XOS production.

Topics: Cajanus; Cellulose; Disaccharides; Glucuronates; Lignin; Oligosaccharides; Polysaccharides; Trisaccharides; Xylans

We have previously described two forms of an endo-β-1,4-xylanase (XynSW2A and XynSW2B) synthesized by thermotolerant Streptomyces sp. SWU10. Here, we describe another xylanolytic enzyme, designated XynSW1. The enzyme was purified to homogeneity from 2 L of culture filtrate. Its apparent molecular mass was 24 kDa. The optimal pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was stable in a wide pH ranges (pH 1-11), more than 80 % of initial activity remained at pH 2-11 after 16 h of incubation at 4 °C and stable up to 50 °C for 1 h. Xylobiose and xylotriose were the major xylooligosaccharides released from oat spelt xylan by the action of XynSW1, indicating of endo-type xylanase. The complete xynSW1 gene contains 1,011 bp in length and encode a polypeptide of 336 with 41 amino acids of signal peptide. The amino acid sequence analysis revealed that it belongs to glycoside hydrolase family 11 (GH11). The mature xynSW1 gene without signal peptide sequence was overexpressed in Pichia pastoris KM71H. The recombinant XynSW1 protein showed higher molecular mass due to the differences in glycosylation levels at the six N-glycosylation sites in the amino acid sequence and exhibited better physicochemical properties than those of the native enzyme including higher optimal temperature (60 °C), and specific activity, but lower optimal pH (4.0). Because of their stability in a wide pH ranges, both of native and recombinant enzymes of XynSW1, may have potential application in several industries including food, textile, biofuel, and also waste treatment.

Topics: Amino Acid Sequence; Cloning, Molecular; Disaccharides; Endo-1,4-beta Xylanases; Gene Expression; Pichia; Streptomyces; Temperature; Trisaccharides

In this study, a process for producing XOS from Sehima nervosum grass was developed. The grass contains 28.1% hemicellulose. NaOH and steam application yielded 98% of original xylan in contrast to 85% by KOH application. Hydrolysis of xylan with commercial xylanase caused breakdown into XOS comprising of xylobiose, xylotriose along with xylose. Response surface model (RSM) revealed highest xylobiose yield (11 g/100g xylan) at pH 5.03, temperature 45.19°C, reaction time 10.11h with enzyme dose 17.41 U. Similarly for maximizing xylotriose yield, ideal hydrolysis conditions were pH 5.11, temperature 40.33°C, reaction time 16.55 h with enzyme dose 13.20 U. A two step process encompassing xylan fractionation and enzymatic hydrolysis enabled XOS production from the S. nervosum grass.

Topics: Alkalies; Disaccharides; Endo-1,4-beta Xylanases; Glucuronates; Hydrogen-Ion Concentration; Hydrolysis; Hydroxides; Oligosaccharides; Poaceae; Potassium Compounds; Sodium Hydroxide; Solubility; Spectroscopy, Fourier Transform Infrared; Temperature; Time Factors; Trisaccharides; Xylans; Xylose

The effects of xylo-oligosaccharides (XOS) and xylose on the hydrolytic activities of cellulases, endoglucanase II (EGII, originating from Thermoascus aurantiacus), cellobiohydrolase I (CBHI, from T. aurantiacus), and cellobiohydrolase II (CBHII, from Trichoderma reesei) on Avicel and nanocellulose were investigated. After the addition of XOS, the amounts of cellobiose, the main product released from Avicel and nanocellulose by CBHI, decreased from 0.78 and 1.37 mg/ml to 0.59 and 1.23 mg/ml, respectively. During hydrolysis by CBHII, the amounts of cellobiose released from the substrates were almost cut in half after the addition of XOS. Kinetic experiments showed that xylobiose and xylotriose were competitive inhibitors of CBHI. The results revealed that the strong inhibition of cellulase by XOS can be attributed to the inhibitory effect of XOS especially on cellobiohydrolase I. The results indicate the necessity to totally hydrolyze xylo-oligosaccharides into the less inhibitory product, xylose, to increasing hydrolytic efficiency.

Topics: Cellulase; Cellulose 1,4-beta-Cellobiosidase; Disaccharides; Enzyme Inhibitors; Glucuronates; Hydrolysis; Kinetics; Oligosaccharides; Thermoascus; Trisaccharides

The xylanolytic bacterium Paenibacillus sp. strain W-61 encodes three extracellular xylanase genes, xyn1, xyn3, and xyn5. In this study, we identified a transcriptional activator required for transcription of the xyn3 gene in strain W-61. The activator, AxyR, contained the highly homologous AraC-type DNA binding domain and required xylobiose, xylotriose, or xylotetraose as cofactor for binding to the xyn3 promoter region.

Topics: AraC Transcription Factor; Bacterial Proteins; Binding Sites; Coenzymes; Disaccharides; Endo-1,4-beta Xylanases; Paenibacillus; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Transcription, Genetic; Transcriptional Activation; Trisaccharides; Xylans

Commercial cellulase complexes produced by cellulolytic fungi contain enzyme activities that are capable of hydrolyzing non-cellulosic polysaccharides in biomass, primarily hemicellulose and pectins, in addition to cellulose. However, xylanase activities detected in most commercial enzyme preparations have been shown to be insufficient to completely hydrolyze xylan, resulting in high xylooligomer concentrations remaining in the hydrolysis broth. Our recent research showed that these xylooligomers are stronger inhibitors of cellulase activity than others have previously established for glucose and cellobiose, making their removal of great importance. In this study, a HPLC system that can measure xylooligomers with degrees of polymerization (DP) up to 30 was applied to assess how Spezyme CP cellulase, Novozyme 188 β-glucosidase, Multifect xylanase, and non-commercial β-xylosidase enzymes hydrolyze different chain length xylooligomers derived from birchwood xylan. Spezyme CP cellulase and Multifect xylanase partially hydrolyzed high DP xylooligomers to lower DP species and monomeric xylose, while β-xylosidase showed the strongest ability to degrade both high and low DP xylooligomers. However, about 10-30% of the higher DP xylooligomers were difficult to be breakdown by cellulase or xylanase and about 5% of low DP xylooligomers (mainly xylobiose) proved resistant to hydrolysis by cellulase or β-glucosidase, possibly due to low β-xylosidase activity in these enzymes and/or the precipitation of high DP xylooligomers.

Topics: Bacteria; beta-Glucosidase; Betula; Cellulase; Disaccharides; Endo-1,4-beta Xylanases; Glycoside Hydrolases; Hydrolysis; Kinetics; Oligosaccharides; Polymerization; Trisaccharides; Xylose

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from decaying sweet gum wood, secretes a multimodular glycohydrolase family GH10 endoxylanase (XynA1) anchored to the cell surface. The gene encoding XynA1 is part of a xylan utilization regulon that includes an aldouronate utilization gene cluster with genes encoding a GH67 alpha-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/beta-xylosidase (XynB). Here we show that this Paenibacillus sp. strain is able to utilize methylglucuronoxylose (MeGAX(1)), an aldobiuronate product that accumulates during acid pretreatment of lignocellulosic biomass, and methylglucuronoxylotriose (MeGAX(3)), the product of the extracellular XynA1 acting on methylglucuronoxylan (MeGAX(n)). The average rates of utilization of MeGAX(n), MeGAX(1), and MeGAX(3) were 149.8, 59.4, and 54.3 microg xylose equivalents.ml(-1).h(-1), respectively, and were proportional to the specific growth rates on the substrates. AguA was active with MeGAX(1) and MeGAX(3), releasing 4-O-methyl-d-glucuronate alpha-1,2 linked to a nonreducing terminal xylose residue. XynA2 converted xylotriose, generated by the action of AguA on MeGAX(3), to xylose and xylobiose. The ability to utilize MeGAX(1) provides a novel metabolic potential for bioconversion of acid hydrolysates of lignocellulosics. The 2.8-fold-greater rate of utilization of polymeric MeGAX(n) than that of MeGAX(3) indicates that there is coupling of extracellular depolymerization, assimilation, and intracellular metabolism, allowing utilization of lignocellulosics with minimal pretreatment. Along with adjacent genes encoding transcriptional regulators and ABC transporter proteins, the aguA and xynA2 genes in the cluster described above contribute to the efficient utilization of aldouronates derived from dilute acid and/or enzyme pretreatment protocols applied to the conversion of hemicellulose to biofuels and chemicals.

Topics: Bacterial Proteins; Disaccharides; Endo-1,4-beta Xylanases; Gene Order; Glucuronidase; Glycosides; Gram-Positive Bacteria; Lignin; Models, Biological; Multigene Family; Polysaccharides; Trisaccharides; Uronic Acids; Xylose

A gene encoding the xylanase of Bacillus subtilis AMX-4 isolated from soil was cloned into Escherichia coli, and the gene product was purified from the cell-free extract of the recombinant strain. The gene, designated xylA, consisted of 639 nucleotides encoding a polypeptide of 213 residues. The deduced amino acid sequence was highly homologous to those of xylanase belonging to glycosyl hydrolase family 11. The molecular mass of the purified xylanase was 23 kDa as estimated by SDS-PAGE. The enzyme had a pH optimum at 6.0-7.0 and a temperature optimum at 50-55 degrees C. Xylanase activity was significantly inhibited by 5 mM Cu2+ and 5 mM Mn2+, and noticeably enhanced by 5 mM Fe2+. The enzyme was active on xylans including arabinoxylan, birchwood xylan, and oat spelt xylan, but it did not exhibit activity toward carboxymethylcellulose or p-nitrophenyl-beta-xylopyranoside. The predominant products resulting from xylan and xylooligosaccharide hydrolysis were xylobiose and xylotriose. The enzyme could hydrolyze xylooligosaccharides larger than xylotriose.

Topics: Bacillus subtilis; Cloning, Molecular; Disaccharides; Endo-1,4-beta Xylanases; Escherichia coli; Genes, Bacterial; Hydrogen-Ion Concentration; Metals, Heavy; Molecular Weight; Recombinant Proteins; Sequence Homology, Amino Acid; Soil Microbiology; Substrate Specificity; Temperature; Trisaccharides; Xylans

Fluorogenic substrates of endo-beta-(1-->4)-xylanases (EXs), 4-methylumbelliferyl beta-glycosides of xylobiose and xylotriose were synthesized from fully acetylated oligosaccharides using the alpha-trichloroacetimidate procedure. A commercially available syrup containing xylose and xylo-oligosaccharides was used as the starting material. Both fluorogenic glycosides were found to be suitable substrates for EXs, particularly for sensitive detection of the enzymes in electrophoretic gels and their in situ localization on sections of fruiting bodies of some plants, such as tomato, potato and eggplant, all of the family Solanaceae.

Topics: Disaccharides; Electrophoresis; Endo-1,4-beta Xylanases; Fluorescent Dyes; Plants; Trisaccharides; Xylose

It is evaluated the effectiveness of the combined action of two highly thermostable enzymes for the hydrolysis of xylans at high temperature in order to produce D-xylose.. Xylans from different sources were hydrolyzed at high degree at 70 degrees C by co-action of a xylanase from the thermophilic bacterium Anoxybacillus flavithermus BC and the novel beta-xylosidase/alpha-arabinosidase from the hyperthermophilic crenarchaeon Sulfolobus solfataricus Oalpha. Beechwood xylan was the best substrate among the xylans tested giving, by incubation only with xylanase, 32.8 % hydrolysis after 4 h. The addition of the beta-xylosidase/alpha-arabinosidase significantly improved the rate of hydrolysis, yielding 63.6% conversion after 4 h incubation, and the main sugar identified was xylose.. This study demonstrates that a significant degree of xylan degradation was reached at high temperature by co-action of the two enzymes. Xylose was obtained as a final product in considerable yield.. Although the xylan represents the second most abundant polysaccharide in nature, it still doesn't have significant utilization for the difficulties encountered in its hydrolysis. Its successful hydrolysis to xylose in only one stage process could make of it a cheap sugar source and could have an enormous economic potential for the conversion of plant biomass into fuels and chemicals.

Topics: Bacillaceae; Chromatography, High Pressure Liquid; Disaccharides; Endo-1,4-beta Xylanases; Glucosidases; Glycoside Hydrolases; Hot Temperature; Hydrolysis; Sulfolobus solfataricus; Trisaccharides; Xylans; Xylose