Aldouronic Acids Research Articles

A GH115 α-glucuronidase structure reveals dimerization-mediated substrate binding and a proton wire potentially important for catalysis.

Xylan is a major constituent of plant cell walls and is a potential source of biomaterials, and the derived oligosaccharides have been shown to have prebiotic effects. Xylans can be highly substituted with different sugar moieties, which pose steric hindrance to the xylanases that catalyse the hydrolysis of the xylan backbone. One such substituent is α-D-glucuronic acid, which is linked to the O2' position of the β-1,4 D-xylopyranoses composing the main chain of xylans. The xylan-specific α-glucuronidases from glycoside hydrolase family 115 (GH115) specifically catalyse the removal of α-D-glucuronic acid (GlcA) or methylated GlcA (MeGlcA). Here, the molecular basis by which the bacterial GH115 member wtsAgu115A interacts with the main chain of xylan and the indirect involvement of divalent ions in the formation of the Michaelis-Menten complex are described. A crystal structure at 2.65 Å resolution of wtsAgu115A originating from a metagenome from an anaerobic digester fed with wastewater treatment sludge was determined in complex with xylohexaose, and Asp303 was identified as the likely general acid. The residue acting as the general base could not be identified. However, a proton wire connecting the active site to the metal site was observed and hence a previous hypothesis suggesting a Grotthuss-like mechanism cannot be rejected. Only a single molecule was found in the asymmetric unit. However, wtsAgu115A forms a dimer with a symmetry-related molecule in the crystal lattice. The xylohexaose moieties of the xylohexaose are recognized by residues from both protomers, thus creating a xylohexaose recognition site at the dimer interface. The dimer was confirmed by analytical size-exclusion chromatography in solution. Kinetic analysis with aldouronic acids resulted in a Hill coefficient of greater than 2, suggesting cooperativity between the two binding sites. Three Ca2+ ions were identified in the wtsAgu115A structures. One Ca2+ ion is of particular interest as it is coordinated by the residues of the loops that also interact with the substrate. Activity studies showed that the presence of Mg2+ or Mn2+ resulted in a higher activity towards aldouronic acids, while the less restrictive coordination geometry of Ca2+ resulted in a decrease in activity.

Non-Specific GH30_7 Endo-β-1,4-xylanase from Talaromyces leycettanus.

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-β-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear β-1,3-β-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched β-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear β-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the −2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.

Cloning, Expression and Characterization of the α-glucuronidase from the Hyperthermophile DictyoglomusturgidumDSM 6724Ô

Conversion of biomass into fermentable sugars is a major requirement for successful and cost-effective biofuels production. The conversion of xylan to sugars requires multiple enzymes including α-glucuronidase. Here we report the cloning, expression, purification and characterization of the α-glucuronidase from Dictyoglomusturgidum(DtuAgu). DtuAgu is an intracellular protein of 685 amino acids and a predicted molecular weight of 79.4 kD. Enzymatic activity was optimum between pH 7.0 and 8.0 and at 85°C. The specific activity of the enzyme was 10 u/mg when measured using mixed aldouronic acids. The specific activity on isolated glucuronoxylan was approximately 20% of the value obtained with xylooligosaccharides. DtuAgu significantly improved xylan conversion to xylose when evaluated using two mixtures of thermostable bacterial enzymes and two sources of xylan. DtuAgu has the potential to be a key player in thermostable enzyme cocktails for the conversion to biomass to biofuels.α

The structural basis of fungal glucuronoyl esterase activity on natural substrates

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an α/β-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.

A novel GH30 xylobiohydrolase from Acremonium alcalophilum releasing xylobiose from the non-reducing end

Xylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase AaXyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates AaXyn30A generates xylobiose as the main product. It was proven that xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. AaXyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear β-1,3-β-1,4-xylan) from which also an isomeric xylotriose Xyl-β-1,3-Xyl-β-1,4-Xyl is formed. Production of xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases.

Glucuronoxylan 3-O-acetylated on uronic acid-substituted xylopyranosyl residues and its hydrolysis by GH10, GH11 and GH30 endoxylanases

Glucuronoxylan selectively 3-O-acetylated on uronic acid-substituted xylopyranosyl residues was prepared by deacetylation of steam explosion-extracted aspenwood acetylglucuronoxylan by the CE6 acetylxylan esterase from Orpinomyces sp. The 3-O-acetylation of MeGlcA-substituted xylopyranosyl residues did not influence the mode of action of GH10, 11 and 30 xylanases, resulting in similar aldouronic acids as are found in alkali-extracted glucuronoxylan hydrolysates. In all three hydrolysates of the selectively acetylated glucuronoxylan, however, 3-O-acetylated aldouronic acids predominated over non-acetylated ones, suggesting that in native aspenwood xylan almost all MeGlcA-substituted Xylp residues are 3-O-acetylated. The results contribute to current knowledge of the mode of action of xylanases and also point to a possibility to produce novel types of xylooligosaccharides. The 3-O-acetylated aldouronic acids, along with the specifically 3-O-acetylated glucuronoxylan, may serve as model substrates for searching for a novel type of esterase able to liberate this MeGlcA-shielded acetyl group. Such esterases are important to improve significantly saccharification yields.

The natural catalytic function of CuGE glucuronoyl esterase in hydrolysis of genuine lignin\u2013carbohydrate complexes from birch

BackgroundGlucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin–carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood.ResultsHere, we show for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan.ConclusionThe data verify the enzyme’s unique ability to catalyze removal of all glucuronoxylan associated with lignin and we propose that this is a direct result of enzymatic cleavage of the ester bonds connecting glucuronoxylan to lignin via 4-O-methyl glucuronoyl-ester linkages. This function appears important for the fungal organism’s ability to effectively utilize all available carbohydrates in lignocellulosic substrates. In bioprocess perspectives, this enzyme is a clear candidate for polishing lignin for residual carbohydrates to achieve pure, native lignin fractions after minimal pretreatment.

Action of different types of endoxylanases on eucalyptus xylan in situ.

Most studies of the mode of action of industrially important endoxylanases have been done on alkali extracted-plant xylan. In just few cases, the native form of the polysaccharide, acetylated xylan, was used as a substrate. In this work action of xylanases belonging to three glycoside hydrolase families, GH10, GH11, and GH30 was investigated on acetylglucuronoxylan directly in hardwood cell walls. Powdered eucalyptus wood was used as xylanase substrate. Enzyme-generated fragments were characterized by TLC, MALDI ToF MS, and NMR spectroscopy. All three xylanases generated from eucalyptus wood powder acetylated xylooligosaccharides. Those released by GH10 enzyme were the shortest, and those released by GH30 xylanase were of the largest diversity. For GH30 xylanase the 4-O-methyl-D-glucuronic acid (MeGlcA) side residues function as substrate specificity determinants regardless the acetylation of the neighboring hydroxyl group. Much simpler xylooligosaccharide patterns were observed when xylanases were applied in combination with carbohydrate esterase family 6 acetylxylan esterase. In the presence of the esterase, all aldouronic acids remained 3-O-acetylated on the xylopyranosyl (Xylp) residue substituted with MeGlcA. The 3-O-acetyl group, in contrast to the acetyl groups of otherwise unsubstituted Xylp residues, does not affect the mode of action of endoxylanases, but contributes to recalcitrance of the acidic xylan fragments. The results confirm importance of acetylxylan esterases in microbial degradation of acetylated hardwood glucuronoxylan. They also point to still unresolved question of efficient enzymatic removal of the 3-O-acetyl group on MeGlcA-substituted Xylp residues negatively affecting the saccharification yields.

Characterization of Thermotoga thermarum DSM 5069 α-Glucuronidase and Synergistic Degradation of Xylan

α-Glucuronidases are capable of breaking down the α-1,2-glycosidic bonds of 4-O-methyl-D-glucuronic acid residues. As an accessory enzyme, α-glucuronidase plays a vital role in xylan degradation. The recombinant α-glucuronidase from Thermotoga thermarum DSM 5069 was heterologously expressed in the Escherichia coli system, purified, and characterized. The purified enzyme exhibited optimal activity toward aldouronic acids at pH 6.5 and 80 °C. It was fairly thermostable and maintained 98% residual activity after incubation at 65 °C for 2.0 h. The kinetic parameters Km, Vmax, and kcat were 3.02 ± 0.16 mM, 88 ± 2 µmol min-1 mg-1, and 117 s-1, respectively. TtAguA had an apparent activation energy of 59.0 kJ/mol. By structure simulation and mutation analyses, Glu288 was identified as the catalytic proton donor, and Asp367 and Glu395 were likely nucleophile bases. The xylan degradation by endoxylanase Xyn10A was enhanced by approximately 10% in the presence of TtAguA.

Expression and Characterization of Glycosyl Hydrolase Family 115 α-Glucuronidase fromScheffersomyces stipitis

Abstract The gene encoding glycosyl hydrolase family 115 α-glucuronidase from Scheffersomyces stipitis (ssagu115) was cloned and expressed in Pichia pastoris strain X33. The recombinant enzyme was purified to homogeneity by Ni-chelation affinity chromatography. The apparent molecular weight of the secreted recombinant enzyme was about 150 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recombinant α-glucuronidase was able to remove 4-O-methylglucuronic acid groups from several polymeric xylans (beech, birch, and oat spelt xylan) as well as xylooligosaccharides (aldotetraouronic acid and aldopentaouronic acid). The enzyme was more active towards xylooligosaccharides than xylans. Substrate inhibition was observed with aldouronic acids at concentrations above 23 mM. The Km values of the enzyme towards aldotetraouronic and aldopentaouronic acids were 8.2 mM (5 mg/mL) and 9 mM (6.5 mg/mL), respectively. For beech and birch xylan, the Km values were 9.5 mg/mL and 35 mg/mL, re...

Novel thermophilic hemicellulases for the conversion of lignocellulose for second generation biorefineries

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and α-glucuronidase from Alicyclobacillus acidocaldarius (GH10-XA and GH67-GA, respectively) and a α-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65°C (3.5 days). In addition, GH67-GC was the most stable α-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-α-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations <0.3μM) and in much less reaction time (2h vs 12h) if compared to other known biotransformations catalyzed by thermophilic enzymes. In addition, remarkably, together with a thermophilic β-xylosidase, they catalyzed the production of xylose from the smart cooking pre-treated biomass of one of the most promising energy crops for second generation biorefineries. We demonstrated that search by the CAZy Data Bank of currently available genomes and detailed enzymatic characterization of recombinant enzymes allow the identification of glycoside hydrolases with novel and interesting properties and applications.

Trichoderma reesei XYN VI--a novel appendage-dependent eukaryotic glucuronoxylan hydrolase.

Expression of a Trichoderma reesei gene coding for a putative GH30 xylanase in Aspergillus oryzae led to isolation and purification of a novel xylanase exhibiting catalytic properties different from those of the previously characterized GH30 xylanase XYN IV of T. reesei. The novel enzyme, named XYN VI, exhibited catalytic properties similar to appendage-dependent GH30 glucuronoxylanases previously recognized only in bacteria. XYN VI showed high specific activity only on xylans or xylooligosaccharides containing 4-O-methyl-D-glucuronic acid or D-glucuronic acid side substituents. The cleavage of the main chain takes place primarily at the second glycosidic linkage from the branch towards the reducing end of the polysaccharides or aldouronic acids. These catalytic properties resemble bacterial GH30 glucuronoxylanases, although the recognition of the uronic acid side chains by XYN VI is apparently based on interaction of the substrate with other amino acids. Moreover, in contrast to bacterial enzymes, XYN VI is also capable of slower but significant cleavage of unsubstituted parts of xylan and acidic xylooligosaccharides. The data point to a great catalytic diversity of xylanases produced by the most extensively studied cellulolytic fungus.

Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose

BackgroundTrichoderma reesei CE16 acetyl esterase (AcE) is a component of the plant cell wall degrading system of the fungus. The enzyme behaves as an exo-acting deacetylase removing acetyl groups from non-reducing end sugar residues. MethodsIn this work we demonstrate this exo-deacetylating activity on natural acetylated xylooligosaccharides using MALDI ToF MS. ResultsThe combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most prominent target is the 3-O-acetyl group at the non-reducing terminal Xylp residues of linear neutral xylooligosaccharides or on aldouronic acids carrying MeGlcA at the non-reducing terminus. Deacetylation of the non-reducing end sugar may involve migration of acetyl groups to position 4, which also serves as substrate of the TrCE16 esterase. ConclusionConcerted action of CtGH10 xylanase, an AcXE and TrCE16 AcE resulted in close to complete deacetylation of neutral xylooligosaccharides, whereas substitution with MeGlcA prevents removal of acetyl groups from only a small fraction of the aldouronic acids. Experiments with diacetyl derivatives of methyl β-d-xylopyranoside confirmed that the best substrate of TrCE16 AcE is 3-O-acetylated Xylp residue followed by 4-O-acetylated Xylp residue with a free vicinal hydroxyl group. General significanceThis study shows that CE16 acetyl esterases are crucial enzymes to achieve complete deacetylation and, consequently, complete the saccharification of acetylated xylans by xylanases, which is an important task of current biotechnology.

Characterization of a recombinant α-glucuronidase from Aspergillus fumigatus

The degradation of xylan requires the action of glycanases and esterases which hydrolyse, in a synergistic fashion, the main chain and the different substituents which decorate its structure. Among the xylanolytic enzymes acting on side-chains are the α-glucuronidases (AguA) (E.C. 3.2.1.139) which release methyl glucuronic acid residues. These are the least studies among the xylanolytic enzymes. In this work, the gene and cDNA of an α-glucuronidase from a newly isolated strain of Aspergillus fumigatus have been sequenced, and the gene has been expressed in Pichia pastoris. The gene is 2523 bp long, has no introns and codes for a protein of 840 amino acid residues including a putative signal peptide of 19 residues. The mature protein has a calculated molecular weight of 91 725 and shows 99 % identity with a putative α-glucuronidase from A. fumigatus A1163. The recombinant enzyme was expressed with a histidine tag and was purified to near homogeneity with a nickel nitriloacetic acid (Ni-NTA) column. The purified enzyme has a molecular weight near 100 000. It is inactive using birchwood glucuronoxylan as substrate. Activity is observed in the presence of xylooligosaccharides generated from this substrate by a family 10 endoxylanase and when a mixture of aldouronic acids are used as substrates. If, instead, family 11 endoxylanase is used to generate oligosaccharides, no activity is detected, indicating a different specificity in the cleavage of xylan by family 10 and 11 endoxylanases. Enzyme activity is optimal at 37 °C and pH 4.5–5. The enzyme binds cellulose, thus it likely possesses a carbohydrate binding module. Based on its properties and sequence similarities the catalytic module of the newly described α-glucuronidase can be classified in family 67 of the glycosyl hydrolases. The recombinant enzyme may be useful for biotechnological applications of α-glucuronidases.

Enzymatically derived aldouronic acids from Cryptomeria japonica arabinoglucuronoxylan

Abstract An arabinoglucuronoxylan was extracted from the holocellulose of sugi ( Cryptomeria japonica ) wood with 10% KOH and subjected to hydrolysis by partially purified xylanase fraction from a commercial cellulase preparation “Meicelase”. Neutral sugars liberated were analyzed by size exclusion chromatography showing the presence of xylooligosaccharides up to xylohexaose. Aldouronic acids liberated were purified by preparative anion exchange chromatography. Their structures were identified by monosaccharide analysis, comparison of their volume distribution coefficients ( Dv s) with those of the authentic samples in anion exchange chromatography and 1 H and 13 C NMR spectroscopy, resulting in the characterization of eight aldouronic acids including acids consisting of two 4- O -Me-α-D-GlcA p residues and 3–5 D-Xyl residues. 1. Fr. 1-S1: (aldohexaouronic acid, MeGlcA 3 Xyl 5 ), O -β-Xyl p -(1 → 4)- O -β-D-Xyl p -(1 → 4)-[ O -(4- O -Me-α-D-GlcA p )-(1 → 2)]- O -β-Xyl p -(1 → 4)- O -β-D-Xyl p -(1 → 4)-D-Xyl 2. Fr. 1-S2: (aldopentaouronic acid, MeGlcA 3 Xyl 4 ), O -β-Xyl p -(1 → 4)-[ O -(4- O -Me-α-D-GlcA p )-(1 → 2)]- O -β-D-Xyl p -(1 → 4)- O -β-Xyl p -(1 → 4)-D-Xyl 3. Fr. 2-S1: (aldotetraouronic acid, MeGlcA 3 Xyl 3 ), O -(4- O -Me-α-D-GlcA p )-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -β-D-Xyl p -(1 → 4)-D-Xyl 4. Fr. 3-S1: (aldotetraouronic acid, GlcA 3 Xyl 3 ), O -(α-D-GlcA p )-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -β-Xyl p -(1 → 4)-D-Xyl, 5. Fr. 4-S1: (aldotriouronic acid, GlcA 2 Xyl 2 ), O -(4- O -Me-α-D-GlcA p )-(1 → 2)- O -β-D-Xyl p -(1 → 4)-D-Xyl 6. Fr. 4-S2: (MeGlc 4 MeGlcA 3 Xyl 5 ), O -β-D-Xyl p -(1 → 4)-[ O -(4- O -Me-α-D-GlcA p )]-(1 → 2)- O -β-D-Xyl p -(1 → 4)-[ O -(4- O -Me-α-D-GlcA p )]-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -β-D-Xyl p -(1 → 4)-D-Xyl 7. Fr. 6-S1: (MeGlcA 4 MeGlcA 3 Xyl 4 ), O -(4- O -Me-α-D-GlcA p )-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -[(4- O -Me-α-D-GlcA p )]-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -β-D-Xyl p -(1 → 4)-D-Xyl 8. Fr. 7-S1: (MeGlcA 3 MeGlc 2 Xyl 3 ), O -(4- O -Me-α-D-GlcA p )-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -[(4- O -Me-α-D-GlcA p )]-(1 → 2)- O -β-D-Xyl p -(1 → 4)-D-Xyl Fr. 4-S2 was a new acidic oligosaccharide. The distribution pattern of these vicinal uronic acid units along the D-xylan chain was discussed.

Structural unit of xylans from sugi (Cryptomeria japonica) and hinoki (Chamaecyparis obtusa)

Arabinoglucuronoxylans (AGXs) isolated from the holocellulose of sugi (Cryptomeria japonica) and hinoki (Chamaecyparis obtusa) contained one 4-O-methyl-d-glucopyranosyluronic acid (4-O-Me-d-GlcAp) residue per 6.2 d-xylopyranose (d-Xylp) residues and one 4-O-Me-d-GlcAp residue per 3.8 d-Xylp residues. These AGXs were subjected to partial acid hydrolysis. Analyses by size exclusion chromatography and electrospray-ionization mass spectroscopy of the neutral sugar fractions in the hydrolysates showed the presence of xylooligosaccharides having a degree of polymerization of 2-8 in addition to d-Xyl, suggesting that the AGXs from sugi and hinoki contained unsubstituted chains consisting of at least eight d-Xyl residues. The acidic sugars in the hydrolysates were separated into two series of aldouronic acids composed of 4-O-Me-d-GlcAp and d-Xylp by ion-exchange chromatography. The first series included aldouronic acids from aldobiouronic acid (4-O-Me-d-GlcAp-Xyl) to aldopentaouronic acids (4-O-Me-d-GlcAp-Xyl4). The second series were aldouronic acids composed of two 4-O-Me-d-GlcAp residues and 2-4 d-Xyl residues. In these acidic sugars, the uronic acid side chains were located on two contiguous d-Xyl residues. These facts indicated that AGXs from sugi and hinoki had a structural unit containing two 4-O-Me-d-GlcAp residues on two contiguous d-Xyl residues as well as AGXs from spruce and larch.

Inverting character of family GH115 α-glucuronidases

α-Glucuronidases of glycoside hydrolase family 115 of the xylose-fermenting yeast Pichia stipitis and wood-destroying fungus Schizophyllum commune liberate 4- O-methyl- d-glucuronic acid residues from aldouronic acids and glucuronoxylan. The specific activities of both enzymes depended on polymerization degree of the acidic xylooligosaccharides and were inhibited by linear β-1,4-xylooligosaccharides. These results suggest interaction of the enzyme with several xylopyranosyl residues of the xylan main chain. Using 1H NMR spectroscopy and reduced aldopentaouronic acid (MeGlcA 3Xyl 4-ol) as a substrate, it was found that both enzymes are inverting glycoside hydrolases releasing 4- O-methyl- d-glucuronic acid (MeGlcA) as its β-anomer.

Enzymatically derived aldouronic acids from Eucalyptus globulus glucuronoxylan

A glucuronoxylan was extracted from the holocellulose of Eucalyputus globulus wood with 10% KOH and subjected to hydrolysis by a commercial cellulase preparation “Meicelase”. Neutral xylooligosaccharides liberated were analyzed by size exclusion chromatography. Aldouronic acids liberated were purified by preparative anion exchange chromatography. Their structures were studied by monosaccharide analysis, comparison of volume distribution coefficients ( Dvs) in anion exchange chromatography with those of the authentic samples, and 1H and 13C NMR spectroscopy, resulting in the characterization of seven aldouronic acids including a novel one containing galactose residue. O-β- d-Xyl p-(1 → 4)-[ O-(4- O-Me-α- d-GlcA p)-(1 → 2)]- O-β- d-Xyl p-(1 → 4)- O-β- d-Xyl p-(1 → 4)- d-Xyl O-(4- O-Me-α- d-GlcA p)-(1 → 2)- O-β- d-Xyl p-(1 → 4)- O-β- d-Xyl p-(1 → 4)- d-Xyl O-(4- O-Me-α- d-GlcA p)-(1 → 2)- O-β- d-Xyl p-(1 → 4)- d-Xyl O-β- d-Xyl p-(1 → 4)- O-β- d-Xyl p-(1→3)- O-α- l-Rha p-(1 → 2)- O-α- l-GalA p-(1 → 4)- d-Xyl O-β- d-Xyl p-(1 → 4)- O-β- d-Xyl p-(1 → 3)- O-α- l-Rha p-(1 → 2)- d-GalA O-β- d-Xyl p-(1 → 3)- O-α- l-Rha p-(1 → 2)- O-α- d-GalA p-(1 → 4)- d-Xyl O-β- d-Gal p-(1 → 2)- O-(4- O-Me-α- d-GlcA p)-(1 → 2)- O-β- d-Xyl p-(1 → 4)- O-β- d-Xyl p-(1 → 4)- d-Xyl. The oligosaccharides liberated provide information on multiplicity of xylanases secreted by Trichoderma viride. The presence of the last aldouronic acid shows a structural feature of E. globulus xylan.

Structures of aldouronic acids liberated from kenaf xylan by endoxylanases from Streptomyces sp.

Streptomyces sp. SK519 was cultivated in the medium containing 0.5% birch wood xylan to produce extracellular xylanase. The xylanase was isolated from the culture supernatant by ammonium sulfate precipitation and partially purified by absorbing on the insoluble xylan. Alkali-extracted kenaf glucuronoylan of which main chain primarily consisting of (1-4)-linked β- D-xylopyranose (Xyl p), some of which carry α-(1-2)-linked 4- O-methylglucopyranosyl acid (MeGlcA p) and glucopyranosyluronic acid (GlcA p) as side chain was digested with the xylanase preparation. At the final stage of digestion, main neutral oligosaccharides were xylotriose (Xyl 3) and xylobiose (Xyl 2) followed by xylotetraose (Xyl 4). Xylose was only formed in a trace amount. As acidic oligosaccharides liberated, following seven aldopentao- and aldotetraouronic acids were isolated in a pure state, respectively, by preparative anion-exchange chromatography. Their structures were determined by analysis of constitutional sugar residues and 1H and 13C NMR spectroscopy. Xyl-β-1,4-[MeGlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-Xyl; [MeGlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-Xyl-β-1,4-Xyl; Xyl-β-1,4-[GlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-Xyl [GlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-Xyl-β-1,4-Xyl [MeGlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-D-Xyl [GlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-Xyl Xyl-β-1,4-Xyl-β-1,3-Rha-α-1,2-GalA-α-1,4-Xyl

Chemical structure of kenaf xylan

Methylation and partial acid hydrolysis of xylans from the bast and core of kenaf ( Hibiscus cannabinus) showed that the main chain of these xylans consists of (1 → 4)-linked β- d-xylopyranosyl (Xyl p) residues, some of which carry a α-1,2-linked 4- O-methyl-glucopyranosyluronic acid (Me-GlcA p) and glucopyranosyluronic acid (GlcA p) residues as side chains. Partial hydrolysis of kenaf xylans afforded two series of aldouronic acids from aldobio- to aldotetraouronic acids. The acids of the first series composed of 4- O-Me- d-GlcA p and d-Xyl p residues: 4- O-Me-GlcA-Xyl 3, 4- O-Me-GlcA-Xyl 2 and 4- O-Me-GlcA-Xyl. The second series composed of d-GlcA p and d-Xyl p: GlcA-Xyl 3, GlcA-Xyl 2 and GlcA-Xyl. In addition to these acids, another aldobiouronic acid, 4- O-(α- d-GalA p)- d-Xyl was found to be present in the partial hydrolysate. The molar ratio of GalA, GlcA, 4- O-Me-GlcA, and Xyl residues was calculated to be 1.0:2.0:9.4:119 for the bast xylan and 1.0:1.3:7.9:99.4 for the core xylan.