Structural diversity of xylans in the cell walls of monocots

Main conclusion

Xylans in the cell walls of monocots are structurally diverse. Arabinofuranose-containing glucuronoxylans are characteristic of commelinids. However, other structural features are not correlated with the major transitions in monocot evolution. Most studies of xylan structure in monocot cell walls have emphasized members of the Poaceae (grasses). Thus, there is a paucity of information regarding xylan structure in other commelinid and in non-commelinid monocot walls. Here, we describe the major structural features of the xylans produced by plants selected from ten of the twelve monocot orders. Glucuronoxylans comparable to eudicot secondary wall glucuronoxylans are abundant in non-commelinid walls. However, the α-d-glucuronic acid/4-O-methyl-α-d-glucuronic acid is often substituted at O-2 by an α-l-arabinopyranose residue in Alismatales and Asparagales glucuronoxylans. Glucuronoarabinoxylans were the only xylans detected in the cell walls of five different members of the Poaceae family (grasses). By contrast, both glucuronoxylan and glucuronoarabinoxylan are formed by the Zingiberales and Commelinales (commelinids). At least one species of each monocot order, including the Poales, forms xylan with the reducing end sequence -4)-β-d-Xylp-(1,3)-α-l-Rhap-(1,2)-α-d-GalpA-(1,4)-d-Xyl first identified in eudicot and gymnosperm glucuronoxylans. This sequence was not discernible in the arabinopyranose-containing glucuronoxylans of the Alismatales and Asparagales or the glucuronoarabinoxylans of the Poaceae. Rather, our data provide additional evidence that in Poaceae glucuronoarabinoxylan, the reducing end xylose residue is often substituted at O-2 with 4-O-methyl glucuronic acid or at O-3 with arabinofuranose. The variations in xylan structure and their implications for the evolution and biosynthesis of monocot cell walls are discussed.

     

Induction and glucose repression of endo-β-xylanase in the yeast Trichosporon cutaneum SL409

Extracellular endo-1,4-β-xylanase (EC 3.2.1.8) synthesis in the yeast Trichosporon cutaneum SL409 is inducible. The enzyme can be induced in washed glucose-grown cells by xylan or xylose. Methyl β-d-xylopyranoside, a synthetic analogue of xylobiose, however, was not an inducer of xylanase in Trichosporon cutaneum SL409. The induction of xylanase by xylan lasted longer and the final total activities were significantly higher compared to the induction by xylose. Xylanase induction was subject to glucose repression.     

Induction of xylanase inAspergillus ochraceus

Aspergillus ochraceus produced xylanase inductively in washed glucose-grown mycelia incubated with methyl β-d-xyloside. The production of xylan endo-l,4-β-xylosidase (xylanase) was 4.2 times greater than that obtained in xylan medium. The inducer was not metabolized and was most active at a concentration of 0.3 g/L with an incubation period of 16 h. The uptake of the inducer by the mycelia seemed to be energy-dependent. Methyl α-d-xyloside repressed xylanase synthesis. Glucose, cyeloheximide, actinomycin D and eAMP were found to inhibit xylanase induction by methyl β-D-xyloside.     

Identification and characterisation of xylanolytic yeasts isolated from decaying wood and sugarcane bagasse in Brazil

In this study, yeasts associated with lignocellulosic materials in Brazil, including decaying wood and sugarcane bagasse, were isolated, and their ability to produce xylanolytic enzymes was investigated. A total of 358 yeast isolates were obtained, with 198 strains isolated from decaying wood and 160 strains isolated from decaying sugarcane bagasse samples. Seventy-five isolates possessed xylanase activity in solid medium and were identified as belonging to nine species: Candida intermedia, C. tropicalis, Meyerozyma guilliermondii, Scheffersomyces shehatae, Sugiyamaella smithiae, Cryptococcus diffluens, Cr. heveanensis, Cr. laurentii and Trichosporon mycotoxinivorans. Twenty-one isolates were further screened for total xylanase activity in liquid medium with xylan, and five xylanolytic yeasts were selected for further characterization, which included quantitative analysis of growth in xylan and xylose and xylanase and β-d-xylosidase activities. The yeasts showing the highest growth rate and cell density in xylan, Cr. laurentii UFMG-HB-48, Su. smithiae UFMG-HM-80.1 and Sc. shehatae UFMG-HM-9.1a, were, simultaneously, those exhibiting higher xylanase activity. Xylan induced the highest level of (extracellular) xylanase activity in Cr. laurentii UFMG-HB-48 and the highest level of (intracellular, extracellular and membrane-associated) β-d-xylosidase activity in Su. smithiae UFMG-HM-80.1. Also, significant β-d-xylosidase levels were detected in xylan-induced cultures of Cr. laurentii UFMG-HB-48 and Sc. shehatae UFMG-HM-9.1a, mainly in extracellular and intracellular spaces, respectively. Under xylose induction, Cr. laurentii UFMG-HB-48 showed the highest intracellular β-d-xylosidase activity among all the yeast tested. C. tropicalis UFMG-HB 93a showed its higher (intracellular) β-d-xylosidase activity under xylose induction and higher at 30 °C than at 50 °C. This study revealed different xylanolytic abilities and strategies in yeasts to metabolise xylan and/or its hydrolysis products (xylo-oligosaccharides and xylose). Xylanolytic yeasts are able to secrete xylanolytic enzymes mainly when induced by xylan and present different strategies (intra- and/or extracellular hydrolysis) for the metabolism of xylo-oligosaccharides. Some of the unique xylanolytic traits identified here should be further explored for their applicability in specific biotechnological processes.     

Characterization of structurally similar neutral and acidic tetrasaccharides obtained from the enzymic hydrolyzate of a 4-O-methyl-D-glucurono-D-xylan

Two similar tetrasaccharides, one neutral and one acidic, were isolated from the products released by the attack of a xylanase on the in situ reduced 4-O-methyl-D-glucurono-D-xylan from aspen (Populus tremuloides). Paper chromatography, gel filtration behavior, methylation followed by reduction, and mass spectrometry showed that the oligosaccharides were O-(4-O-methyl-α-D-glucopyranosyluronic acid)-(1→2)-D-xylotriose and-O-(4-O-methyl-α-D-glucopyranosyluronic acid)-(1→2)-D-xylotriose. Independent of the acidic or neutral substituent on the present xylan chain, the enzymic cleavage led preferentially to oligosaccharides substituted at the nonreducing end. The existence, in wood, of a few uronic acid substituents of the D-xylan in the esterified form was confirmed, and their linkage to lignin postulated.     

The synthesis and degradation of benzyl 4,6-O-benzylidene-2,3-dideoxy-3-C-ethyl-2-C-hydroxymethyl-α-d-glucopyranoside and -mannopyranoside

The use of carbohydrates for establishing, by synthesis, the absolute configuration of branched aliphatic alcohols is demonstrated by the synthesis and degradation of carbohydrate derivatives that contain two branch points. Benzyl 4,6-O-benzylidene-2,3-dideoxy-3-C-ethyl-2-C-hydroxymethyl-α-d-glucopyranoside (23) and -mannopyranoside (24) were formed from benzyl 2,3-anhydro-4,6-O-benzylidene-α-d-mannopyranoside (17) by a reaction sequence that involved ring-opening with ethylmagnesium chloride, oxidation, epimerisation, methylenation, and hydroboronation. The gluco isomer 23 was converted into (+)-(R)-2,3-bisacetoxymethylpentyl acetate (1) by sequential hydrogenolysis, borohydride reduction, periodate oxidation, borohydride reduction, and acetylation. The synthesis of 1 provides confirmatory evidence for the absolute configuration of the alkaloid pilocarpine (2). Unidentified products, and not the expected free-sugars, were obtained by acidic hydrolysis of methyl 4,6-O-benzylidene-2,3-dideoxy-3-C-ethyl-2-C-hydroxymethyl-α-d-glucopyranoside (8) and -mannopyranoside (9). Convenient syntheses of benzyl α-d-glucopyranoside derivatives are described.     

Secreted xylanase XynA mediates utilization of xylan as sole carbon source in <Emphasis Type="Italic">Candida utilis</Emphasis>

The fodder yeast Candida utilis is able to use xylose mono- and oligomers as sources of carbon but not the abundant polymer xylan. C. utilis transformants producing the Penicillium simplicissimum xylanase XynA were constructed using expression vectors encoding fusions of the Saccharomyces cerevisiae Mfα1 pre-pro secretion leader to XynA. The Mfα1-XynA fusion was efficiently processed in transformants and XynA was secreted almost quantitatively into the culture medium. Secreted XynA was enzymatically active and allowed transformants to grow on xylan as the sole carbon source. Addition of a second expression unit for the heterologous green fluorescent protein (GFP) generated C. utilis transformants, which showed intracellular GFP fluorescence during growth on xylan. The results suggest that xylanase-producing C. utilis is suited as a cost-effective host organism for heterologous protein production and for other biotechnical applications.     

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K _m and V _max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.     

Syntheses of model oligosaccharides of biological significance.Synthesis of methyl 3,6-DI-O-(α-d-mannopyranosyl)-α-d-mannopyranoside and the corresponding mannobiosides

Methyl 2-O-allyl-4,6-O-benzylidene-3-O-(2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl)-α-d-mannopyranoside(12) was prepared in 90 % yield by Helferich glycosylation of methyl 2-O-allyl-4,6-O-benzylidene-α-d-mannopyranoside (9) with tetra-O-acetyl-α-d-mannopyranosyl bromide (11). Removal of the benzylidene group and second Helferich glycosylation with 11 led to methyl 2-O-allyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl)-α-d-mannopyranoside (14) which, after deallylation and Zemplén deacetylation, gave the title compound 5. The disaccharides methyl 3-O-(α-d-mannopyranosyl)-α-d-mannopyranoside (7) and methyl 6-O-(α-d-mannopyranosyl)-α-d-mannopyranoside (6) have also been synthesized. Complete assignments of the ¹H-n.m.r. spectra of the compounds 5, 6, and 7 are given.     

Flavonoid glycosides from the aerial parts of Curcuma comosa

Four new flavonoid glycosides, curcucomosides A–D (1–4), three known flavonoid glycosides, 5–7, and four known diarylheptanoids, 8–11, were isolated from the ethanol extract of the aerial parts of Curcuma comosa. The structures of the new compounds were established as rhamnazin 3-O-α-l-arabinopyranoside (1), rhamnocitrin 3-O-α-l-arabinopyranoside (2), rhamnazin 3-O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabinopyranoside (3), and rhamnocitrin 3-O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabinopyranoside (4) by spectroscopic analysis and chemical reactions whereas those of the known compounds were identified by spectral comparison with those of the reported values.     

Production, Purification, and Characterization of a Xylooligosaccharides-forming Xylanase from High-butanol-producing Strain Clostridium sp. BOH3

An endo-acting xylanase is isolated from the culture medium of Clostridium sp. BOH3 when xylan, glucose, xylose, or sugarcane bagasse hydrolysate (SBH) is used as a carbon source. Crude xylanase is purified by using an anionic Q-column with a yield of 39 %. The pure xylanase has a molecular weight of 35.8 kDa, and it shows optimal activity at pH 5 and 60 °C. When beechwood xylan is used as a substrate, this xylanase liberates short oligosaccharides (XOS) predominantly, ranging from xylobiose (X2) to xylopentaose (X5). However, no xylose can be detected, suggesting that this is an endo-β-1,4-xylanase. Kinetic study of this xylanase reveals that K _m and V _max are 1.36 mg/ml and 212 μmol/(min. mg protein), respectively. On the basis of amino acid sequence, this enzyme shows homology to xylanase (xynb) from Clostridium acetobutylicum ATCC 824, but this enzyme has several distinctive characteristics. For example, its activity can be enhanced with the addition of divalent metal ions, and it produces XOS exclusively when xylan is used as a substrate. These unique characteristics suggest that this is a new enzyme.     

Production of thermostable xylanase by a thermophilic fungus,Thermoascus aurantiacus

Twenty-one strains of thermophilic fungi in the Forintek culture collection were screened for their production of xylanolytic (and cellulolytic) enzymes in both solid and aqueous media containing various hemicellulosic and cellulosic substrates. Thermoascus aurantiacus strain C436 was selected as the best producer of extracellular xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) enzymes. High xylanase activity was detected in fungal culture filtrates even when realistic lignocellulosic residues (including steam-exploded aspenwood and untreated aspenwood sawdust) were used as substrates. Maximum xylanase activity (575.9 U ml⁻¹) was detected in cultures grown in Vogel's medium containing oat-spelt xylan. The xylanase activity exhibited a temperature optimum of 75°C and pH optimum around 5.0. The half-lives of the xylanase activity at 70 and 60°C were 1.5 h and 4 days, respectively. Over 90% of the xylanase activity was retained after 12 weeks at 50°C. Crude culture filtrates concentrated by membrane ultrafiltration could effectively hydrolyse xylan and steam-exploded aspenwood hemicellulose to release near theoretical yields of low molecular weight pentose oligomers.     

Synthesis of 8-(hydroxyalkyl)adenines

Treatment of methyl 4,6-O-benzylidene-2-O-p-tolylsulfonyl-α-D-ribo-hexopyranosid-3-ulose (1) with triethylamine-methanol at reflux temperature yields methyl 2,3-anhydro-4,6-O-benzylidene-3-methoxy-α-D-allopyranoside (2), a derivative (3) of 3-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, and methyl 4,6-O-benzylidene-α-D-ribo-hexopyranosid-3-ulose dimethyl acetal (4). The reaction of methyl 4,6-O benzylidene-3-O-p-tolylsulfonyl-α-D-arabino-hexopyranosid-2-ulose (12) with triethylamine-methanol afforded methyl 4,6-O-benzylidene-α-D-ribo-hexopyranosid-2-ulose dimethyl acetal (19) and methyl 2,3-anhydro-4,6-O-benzylidene-2-methoxy-α-D-allopyranoside (20); from the reaction of the β-D anomer (13) of 12, methyl 4,6-O-benzylidene-β-D-ribo-hexopyranosid-2-ulose dimethyl acetal (21) was isolated. Syntheses of the α-keto toluene-p-sulfonates 12 and 13 are described. Mechanisms for the formation of the compounds isolated from the reactions with triethylamine-methanol are proposed.     

Practical synthesis of O-β-d-mannopyranosyl-, O-α-d-mannopyranosyl-, and O-β-d-glucopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→3)-d-galactoses

The koenigs-Knorr glycosylation of 4,6-O-ethylidene-1,2-O-isopropylidene-3-O-(2,3-O-isopropylidene-α-l-rhamnopyranosyl)-α-d-galactopyranose (3) by 4,6-di-O-acetyl-2,3-O-carbonyl-α-d-mannopyranosyl bromide (10), as well as Helferich glycosylations of 3 by tetra-O-acetyl-α-d-mannopyranosyl and -α-d-glucopyranosyl bromides, proceeded smoothly to give high yields of trisaccharide derivatives (12, 16, and 17). An efficient procedure for the transformation of 12, 16, and 17 into the α-deca-acetates of the respective trisaccharides has been developed. Zemplén de-acetylation then afforded the title trisaccharides in yields of 53, 52, and 62 %, respectively, from 3. A new route to 1,4,6-tri-O-acetyl-2,3-O-carbonyl-α-d-mannopyranose is suggested.     

The synthesis of 5-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-β-D-glucofuranose

Condensation of dimeric 3,4,6-tri-O-acetyl-2-deoxy-2-nitroso-α-D-glucopyranosyl chloride (1) with 1,2-O-isopropylidene-α-D-glucofuranurono-6,3-lactone (2) gave 1,2-O-isopropylidene-5-O-(3,4,6-tri-O-acetyl-2-deoxy-2-hydroxyimino-α-D-arabino-hexopyranosyl)-α-D-glucofuranurono-6,3-lactone (3). Benzoylation of the hydroxyimino group with benzoyl cyanide in acetonitrile gave 1,2-O-isopropylidene-5-O-(3,4,6-tri-O-acetyl-2-benzoyloxyimino-2-deoxy-α-D-arabino-hexopyranosyl)-α-D-glucofuranurono-6,3-lactone (4). Compound 4 was reduced with borane in tetrahydrofuran, yielding 5-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-1,2-O-isopropylidene-α-D-glucofuranose (5), which was isolated as the crystalline N-acetyl derivative (6). After removal of the isopropylidene acetal, the pure, crystalline title compound (10) was obtained.     

The synthesis of 3-amino-3-deoxy-α-d-glucopyranosyl α-d-glucopyranoside (3-amino-3-deoxy-α,α-trehalose

The preparation of 2,3-di-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranosyl-2-O-benzoyl-4,6-O-benzylidene-α-d-ribo-hexopyranosid-3-ulose (3) from 4,6:4′,6′-di-O-benzylidene-α,α-trehalose (1) via the 2,3,2′-tribenzoate 2 has been improved. Reduction of 3 with sodium borohydride gave 2-O-benzoyl-4,6-O-benzylidene-α-d-allopyranosyl 2,3-di-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside (4), which was converted into the methanesulfonate 5 and trifluoromethanesulfonate 6. Displacement of the sulfonic ester group in 6 with lithium azide was very facile and afforded a high yield of 3-azido-2-O-benzoyl-4,6-O-benzylidene-3-deoxy-α-d-glucopyranosyl 2,3-di-O-benzoyl-4,6-O-benzylidene-α-d-glycopyranoside (7), whereas similar displacement in 5 proceeded sluggishly, giving a lower yield of 7 together with an unsaturated disaccharide (8). The azido sugar 7 was converted by conventional reactions into the analogous 2,3,2′-triacetate 9, the corresponding 2,3,2′-triol 10, and deprotected 3-azido-3-deoxy-α-d-glucopyranosyl α-d-glucopyranoside (11). Hydrogenation of 11 over Adams' catalyst furnished crystalline 3-amino-3-deoxy-α,α-trehalose hydrochloride (12), the overall yield from 3 being 35%.     

Synthesis of 4-O-α-d-galactopyranosyl-l-rhamnose and 4-O-α-d-galactopyranosyl-2-O-β-glucopyranosyl-l-rhamnose using dioxolane-type benzylidene acetals as temporary protecting-groups

The Halide ion-catalysed reaction of benzyl exo-2,3-O-benzylidene-α-l-rhamnopyranoside with tetra-O-benzyl-α-d-galactopyranosyl bromide and hydrogenolysis of the exo-benzylidene group of the product 2 gave benzyl 3-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-α-d-galactopyranosyl)-α-l-rhamnopyranoside (6). Compound 2 was converted into 4-O-α-d-galactopyranosyl-l-rhamnose. The reaction of 6 with tetra-O-acetyl-α-d-glucopyranosyl bromide and removal of the protecting groups from the product gave 4-O-α-d-galactopyranosyl-2-O-β-d-glucopyranosyl-l-rhamnose.     

Synthesis of 5-O-α-and-β-d-glucopyranosyl-d-glucofuranose and 5-O-α-d-glucopyranosyl-d-fructopyranose (leucrose)

Reaction of 1,2-O-cyclopentylidene-α-d-glucofuranurono-6,3-lactone (2) with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide (1) gave 1,2-O-cyclopentylidene- 5-O-(2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl)-α-d-glucofuranurono-6,3-lactone (3, 45%) and 1,2-O-cyclopentylidene-5-O-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-α-d-glucofuranurono-6,3-lactone (4, 38%). Reduction of 3 and 4 with lithium aluminium hydride, followed by removal of the cyclopentylidene group, afforded 5-O-α-(9) and -β-d-glucopyranosyl-d-glucofuranose (12), respectively. Base-catalysed isomerization of 9 yielded crystalline 5-O-α-d-glucopyranosyl-d-fructopyranose (leucrose, 53%).     

Chemo-sensitizing activity of natural cadinanes from Heterotheca inuloides in human uterine sarcoma cells and their in silico interaction with ABC transporters

Sensitizing activities exerted by 3,4-dihydro-7-hydroxycadalene (1), rac-3,7-dihydroxy-3(4H)-isocadalen-4-one (4) and (1R,4R)-4H-1,2,3,4-tetrahydro-1-hydroxycadalen-15-oic acid (9), the major cadinanes isolated from Heterotheca inuloides, towards multidrug-resistant MES-SA/MX2 and parental MES-SA epithelial human uterine sarcoma cell lines were evaluated. We also evaluated the in silico interactions (expressed as ΔG_binding in kcal/mol) of cadinanes 1, 4 and 9 in an in vitro assay, and also tested several structurally related natural compounds with the multidrug resistance protein (MDR1, P-glycoprotein), human multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP) structures as pharmacological targets using AutoDock and AutoDock Vina. Compound 1 potentiated the cytotoxicity of doxorubicin and mitoxantrone drugs in resistant MES-SA/MX2 cells, compared to cells treated with each drug alone. Compound 1 could reverse the resistance to doxorubicin 12.44 fold at a concentration of 5 μM. It also re-sensitized cells to mitoxantrone 3.94 fold. Hence, compound 1 may be considered as a potential chemosensitizing agent to overcome multidrug resistance in cancer. The docking analysis suggested that there are interactions between cadinanes from H. inuloides and MDR1, MRP1, and BCRP proteins mainly through π-π interactions and hydrogen bonds.