Green Synthesis of Antileishmanial and Antifungal Silver Nanoparticles Using Corn Cob Xylan as a Reducing and Stabilizing Agent.

Corn cob is an agricultural byproduct that produces an estimated waste burden in the thousands of tons annually, but it is also a good source of xylan, an important bioactive polysaccharide. Silver nanoparticles containing xylan (nanoxylan) were produced using an environmentally friendly synthesis method. To do this, we extracted xylan from corn cobs using an ultrasound technique, which was confirmed by both chemical and NMR analyses. This xylan contained xylose, glucose, arabinose, galactose, mannose, and glucuronic acid in a molar ratio of 50:21:14:9:2.5:2.5, respectively. Nanoxylan synthesis was analyzed using UV-vis spectroscopy at kmax = 469 nm and Fourier transform infrared spectroscopy (FT-IR), which confirmed the presence of both silver and xylan in the nanoxylan product. Dynamic light scattering (DLS) and atomic force microscopy (AFM) revealed that the nanoxylan particles were ~102.0 nm in size and spherical in shape, respectively. DLS also demonstrated that nanoxylan was stable for 12 months and coupled plasma optical emission spectrometry (ICP-OES) showed that the nanoxylan particles were 19% silver. Nanoxylan reduced Leishmania amazonensis promastigote viability with a half maximal inhibitory concentration (IC50) value of 25 µg/mL, while xylan alone showed no effective. Additionally, nanoxylan exhibited antifungal activity against Candida albicans (MIC = 7.5 µg/mL), C. parapsilosis (MIC = 7.5 µg/mL), and Cryptococcus neoformans (MIC = 7.5 µg/mL). Taken together, these data suggest that it is possible to synthesize silver nanoparticles using xylan and that these nanoxylan exert improved antileishmanial and antifungal activities when compared to the untreated polysaccharide or silver nitrate used for their synthesis. Thus, nanoxylan may represent a promising new class of antiparasitic agents for use in the treatment of these microorganisms.

Xyloglucan in the primary cell wall: assessment by FESEM, selective enzyme digestions and nanogold affinity tags.

Xyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load-bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high-resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase-digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan-specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold-conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan-depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.

Xyloglucan is released by plants and promotes soil particle aggregation.

Soil is a crucial component of the biosphere and is a major sink for organic carbon. Plant roots are known to release a wide range of carbon-based compounds into soils, including polysaccharides, but the functions of these are not known in detail. Using a monoclonal antibody to plant cell wall xyloglucan, we show that this polysaccharide is secreted by a wide range of angiosperm roots, and relatively abundantly by grasses. It is also released from the rhizoids of liverworts, the earliest diverging lineage of land plants. Using analysis of water-stable aggregate size, dry dispersion particle analysis and scanning electron microscopy, we show that xyloglucan is effective in increasing soil particle aggregation, a key factor in the formation and function of healthy soils. To study the possible roles of xyloglucan in the formation of soils, we analysed the xyloglucan contents of mineral soils of known age exposed upon the retreat of glaciers. These glacial forefield soils had significantly higher xyloglucan contents than detected in a UK grassland soil. We propose that xyloglucan released from plant rhizoids/roots is an effective soil particle aggregator and may, in this role, have been important in the initial colonization of land.

Towards enzymatic breakdown of complex plant xylan structures: State of the art.

Significant progress over the past few years has been achieved in the enzymology of microbial degradation and saccharification of plant xylan, after cellulose being the most abundant natural renewable polysaccharide. Several new types of xylan depolymerizing and debranching enzymes have been described in microorganisms. Despite the increasing variety of known glycoside hydrolases and carbohydrate esterases, some xylan structures still appear quite recalcitrant. This review focuses on the mode of action of different types of depolymerizing endoxylanases and their cooperation with ß-xylosidase and accessory enzymes in breakdown of complex highly branched xylan structures. Emphasis is placed on the enzymatic hydrolysis of alkali-extracted deesterified polysaccharide as well as acetylated xylan isolated from plant cell walls under non-alkaline conditions. It is also shown how the combination of selected endoxylanases and debranching enzymes can determine the nature of prebiotic xylooligosaccharides or lead to complete hydrolysis of the polysaccharide. The article also highlights the possibility for discovery of novel xylanolytic enzymes, construction of multifunctional chimeric enzymes and xylanosomes in parallel with increasing knowledge on the fine structure of the polysaccharide.

Molecular basis of substrate recognition and specificity revealed in family 12 glycoside hydrolases.

Fungal GH12 enzymes are classified as xyloglucanases when they specifically target xyloglucans, or promiscuous endoglucanases when they exhibit catalytic activity against xyloglucan and ß-glucan chains. Several structural and functional studies involving GH12 enzymes tried to explain the main patterns of xyloglucan activity, but what really determines xyloglucanase specificity remains elusive. Here, three fungal GH12 enzymes from Aspergillus clavatus (AclaXegA), A. zonatus (AspzoGH12), and A. terreus (AtEglD) were studied to unveil the molecular basis for substrate specificity. Using functional assays, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that three main regions are responsible for substrate selectivity: (i) the YSG group in loop 1; (ii) the SST group in loop 2; and (iii) loop A3-B3 and neighboring residues. Functional assays and sequence alignment showed that while AclaXegA is specific to xyloglucan, AtEglD cleaves ß-glucan, and xyloglucan. However, AspzoGH12 was also shown to be promiscuous contrarily to a sequence alignment-based prediction. We find that residues Y111 and R93 in AtEglD harbor the substrate in an adequate orientation for hydrolysis in the catalytic cleft entrance and that residues Y19 in AclaXegA and Y30 in AspzoGH12 partially compensate the absence of the YSG segment, typically found in promiscuous enzymes. The results point out the multiple structural factors underlying the substrate specificity of GH12 enzymes. Biotechnol. Bioeng. 2016;113: 2577-2586. © 2016 Wiley Periodicals, Inc.

Specific enzymatic tailoring of wheat arabinoxylan reveals the role of substitution on xylan film properties.

To increase understanding of the applicability of agro biomass by-products as biodegradable film formers, the effect of wheat arabinoxylan (WAX) fine structure on film properties was studied by applying specific enzyme modifications. WAX was selectively modified to mimic the natural variations of different arabinoxylans, particularly the degree of mono and disubstitution of α-L-arabinofuranosyl (Araf) units in ß-D-xylopyranosyl (Xylp) backbone residues. The resulting modified WAX samples had similar arabinose-to-xylose (Ara/Xyl) ratios, but they differed in the number of unsubstituted Xylp units. The substitution of WAX was found to affect, in particular, tensile strength, crystallinity, and oxygen permeability properties of the films, as statistically significant decreases in tensile strength and oxygen permeability took place after WAX de-branching. An increase in the number of unsubstituted Xylp units decreased the temperature of relaxation of small-scale molecular motions of WAX (ß-relaxation) and increased the degree of crystallinity of the films.

Spatial and temporal variability of xylan distribution in differentiating secondary xylem of hybrid aspen.

Xylans occupy approximately one-third of the cell wall components in hardwoods and their chemical structures are well understood. However, the microdistribution of xylans (O-acetyl-4-O-methylglucuronoxylans, AcGXs) in the cell wall and their correlation with functional properties of cells in hardwood xylem is poorly understood. We demonstrate here the spatial and temporal distribution of xylans in secondary xylem cells of hybrid aspen using immunolocalization with LM10 and LM11 antibodies. Xylan labeling was detected earliest in fibers at the cell corner of the S1 layer, and then later in vessels and ray cells respectively. Fibers showed a heterogeneous labeling pattern in the mature cell wall with stronger labeling of low substituted xylans (lsAcGXs) in the outer than inner cell wall. In contrast, vessels showed uniform labeling in the mature cell wall with stronger labeling of lsAcGXs than fibers. Xylan labeling in ray cells was detected much later than that in fibers and vessels, but was also detected at the beginning of secondary cell wall formation as in fibers and vessels with uniform labeling in the cell wall regardless of developmental stage. Interestingly, pit membranes including fiber-, vessel- and ray-vessel pits showed strong labeling of highly substituted xylans (hsAcGXs) during differentiation, although this labeling gradually disappeared during pit maturation. Together our observations indicate that there are temporal and spatial variations of xylan deposition and chemical structure of xylans between cells in aspen xylem. Differences in xylan localization between aspen (hardwood) and cedar (softwood) are also discussed.

The peroxidase/H2O2 system as a free radical-generating agent for gelling maize bran arabinoxylans: rheological and structural properties.

The oxidative gelation of maize bran arabinoxylans (MBAX) using a peroxidase/H(2)O(2) system as a free radical-generating agent was investigated. The peroxidase/H(2)O(2) system led to the formation of dimers and trimer of ferulic acid as covalent cross-link structures in the MBAX network. MBAX gels at 4% (w/v) presented a storage modulus of 180 Pa. The structural parameters of MBAX gels were calculated from swelling experiments. MBAX gels presented a molecular weight between two cross-links (Mc), a cross-linking density (ρ(c)) and a mesh size (x) of 49 × 103 g/mol, 30 × 10-6 mol/cm3 and 193 nm, respectively.

Characterization of a new α-L: -arabinofuranosidase from Penicillium sp. LYG 0704, and their application in lignocelluloses degradation.

A gene (arf) encoding an α-L: -arabinofuranosidase (ARF) that hydrolyzes arabinose substituted on xylan was isolated from Penicillium sp. The gene was predicted to encode 339 amino acid residues showing 71-75% homology to GH family 54. E. coli expressed ARF showed optimal activity at 50°C and pH 5-6 on wheat arabinoxylan. The hydrolysis activities on oat spelt xylan by ARF and xylanase were 1.67-fold higher than that of xylanase alone. The synergistic effects of ARF and commercial enzymes (xylanase and cellulase) on popping-pretreated rice straw were 1.15-1.51-fold higher amounts of sugars released in the [ARF + xylanase + cellulase] mixture than in the mixtures [ARF + xylanase], [ARF + cellulase], and [xylanase + cellulase]. Moreover, the liberation of arabinose by ARF was enhanced 2.1-2.9-fold in a reaction with xylanase and cellulase as compared with [xylanase + cellulase] and ARF alone.

Occurrence of xylan and mannan polysaccharides and their spatial relationship with other cell wall components in differentiating compression wood tracheids of Cryptomeria japonica.

Compression wood (CW) tracheids have different cell wall components than normal wood (NW) tracheids. However, temporal and spatial information on cell wall components in CW tracheids is poorly understood. We investigated the distribution of arabino-4-O-methylglucuronoxylans (AGXs) and O-acetyl-galactoglucomannans (GGMs) in differentiating CW tracheids. AGX labeling began to be detected in the corner of the S(1) layer at the early S(1) formation stage. Subsequently, the cell corner middle lamella (ccML) showed strong AGX labeling when intercellular spaces were not fully formed. AGX labeling was uniformly distributed in the S(1) layer, but showed uneven distribution in the S(2) layer. AGX labeling was mainly detected in the inner S(2) layer after the beginning of the helical cavity formation. The outer S(2) layer showed almost no labeling of low substituted AGXs. Only a very small amount of high substituted AGXs was distributed in the outer S(2) layer. These patterns of AGX labeling in the S(2) layer opposed the lignin and ß-1-4-galactan distribution in CW tracheids. GGM labeling patterns were almost identical to AGX labeling in the early stages of CW tracheids, and GGM labeling was detected in the entire S(2) layer from the early S(2) formation stage of CW tracheids with some spatial differences in labeling density depending on developmental stage. Compared with NW tracheids, CW tracheids showed significantly different AGX distributions in the secondary cell wall but similar GGM labeling patterns. No significant differences were observed in labeling after delignification of CW tracheids.

NaCl effect on the distribution of wall ingrowth polymers and arabinogalactan proteins in type A transfer cells of Medicago sativa Gabès leaves.

We studied the distribution of wall ingrowth (WI) polymers by probing thin sections of companion cells specialized as transfer cells in minor veins of Medicago sativa cv Gabès blade with affinity probes and antibodies specific to polysaccharides and glycoproteins. The wall polymers in the controls were similar in WIs and in the primary wall but differently distributed. The extent of labeling in these papillate WIs differed for JIM5 and JIM7 homogalacturonans but was in the same range for LM5 and LM6 rhamnogalacturonans and xyloglucans. These data show that WI enhancement probably requires arabinogalactan proteins (JIM8) mainly localized on the outer part of the primary wall and WIs. By comparison, NaCl-treated plants exhibited cell wall polysaccharide modifications indicating (1) an increase in unesterified homogalacturonans (JIM5), probably implicated in Na(+) binding and/or polysaccharide network interaction for limiting turgor variations in mesophyll cells; (2) enhancement of the xyloglucan network with an accumulation of fucosylated xyloglucans (CCRC-M1) known to increase the capacity of cellulose binding; and (3) specific recognition of JIM8 arabinogalactan proteins that could participate in both wall enlargement and cohesion by increasing the number of molecular interactions with the other polymers. In conclusion, the cell wall polysaccharide distribution in enlarged WIs might (1) participate in wall resistance to sequestration of Na(+), allowing a better control of hydric homeostasis in mesophyll cells to maintain metabolic activity in source leaves, and (2) maintain tolerance of M. sativa to NaCl.

Hydrolytic properties of a hybrid xylanase and its parents.

The hydrolytic properties of a hybrid xylanase (ATx) and its parents (reAnxA and reTfxA) were studied using xylans and xylooligosaccharides as substrates. Analysis of reaction mixtures by high-performance liquid chromatograph revealed that xylotriose (X3) was the main product released from birchwood xylan and wheat bran insoluble xylan by ATx and reAnxA, respectively. Xylobiose (X2) was the main product separately released from birchwood xylan and wheat bran insoluble xylan by reTfxA. Xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolyzed by ATx, which showed no activity on X2 and X3. Therefore, X4 might be the minimum oligomer hydrolyzed by ATx. X2-X6 could be hydrolyzed by reAnxA and reTfxA, respectively. All of ATx, reAnxA, and reTfxA showed transglycosylation activity.

The reb1-1 mutation of Arabidopsis. Effect on the structure and localization of galactose-containing cell wall polysaccharides.

The Arabidopsis (Arabidopsis thaliana) root epidermal bulger1-1 (reb1-1) mutant (allelic to root hair defective1 [rhd1]) is characterized by a reduced root elongation rate and by bulging of trichoblast cells. The REB1/RHD1 gene belongs to a family of UDP-D-Glucose 4-epimerases involved in the synthesis of D-Galactose (Gal). Our previous study showed that certain arabinogalactan protein epitopes were not expressed in bulging trichoblasts of the mutant. In this study, using a combination of microscopical and biochemical methods, we have investigated the occurrence and the structure of three major Gal-containing polysaccharides, namely, xyloglucan (XyG), rhamnogalacturonan (RG)-I, and RG-II in the mutant root cell walls. Our immunocytochemical data show that swollen trichoblasts were not stained with the monoclonal antibody CCRC-M1 specific for alpha-L-Fucp-(1-->2)-beta-D-Galp side chains of XyG, whereas they were stained with anti-XyG antibodies specific for XyG backbone. In addition, analysis of a hemicellulosic fraction from roots demonstrates the presence of two structurally different XyGs in reb1-1. One is structurally similar to wild-type XyG and the other is devoid of fuco-galactosylated side chains and has the characteristic of being insoluble. Similar to anti-XyG antibodies, anti-bupleuran 2IIC, a polyclonal antibody specific for galactosyl epitopes associated with pectins, stained all root epidermal cells of both wild type and reb1-1. Similarly, anti-RG-II antibodies also stained swollen trichoblasts in the mutant. In addition, structural analysis of pectic polymers revealed no change in the galactosylation of RG-I and RG-II isolated from reb1-1 root cells. These findings demonstrate that the reb1-1 mutation affects XyG structure, but not that of pectic polysaccharides, thus lending support to the hypothesis that biosynthesis of Gal as well as galactosylation of complex polysaccharides is regulated at the polymer level.

Arabinoxylan and (1-->3),(1-->4)-beta-glucan deposition in cell walls during wheat endosperm development.

Arabinoxylans (AX) and (1-->3),(1-->4)-beta-glucans are major components of wheat endosperm cell walls. Their chemical heterogeneity has been described but little is known about the sequence of their deposition in cell walls during endosperm development. The time course and pattern of deposition of the (1-->3) and (1-->3),(1-->4)-beta-glucans and AX in the endosperm cell walls of wheat (Triticum aestivum L. cv. Recital) during grain development was studied using specific antibodies. At approximately 45 degrees D (degree-days) after anthesis the developing walls contained (1-->3)-beta-glucans but not (1-->3),(1-->4)-beta-glucans. In contrast, (1-->3),(1-->4)-beta-glucans occurred widely in the walls of maternal tissues. At the end of the cellularization stage (72 degrees D), (1-->3)-beta-glucan epitopes disappeared and (1-->3),(1-->4)-beta-glucans were found equally distributed in all thin walls of wheat endosperm. The AX were detected at the beginning of differentiation (245 degrees D) in wheat endosperm, but were missing in previous stages. However, epitopes related to AX were present in nucellar epidermis and cross cells surrounding endosperm at all stages but not detected in the maternal outer tissues. As soon as the differentiation was apparent, the cell walls exhibited a strong heterogeneity in the distribution of polysaccharides within the endosperm.

Morphology and gelation of thermosensitive xyloglucan hydrogels.

Galactose modified xyloglucan is a thermally reversible hydrogel that is increasingly used in the biomedical field due to the ease of altering the gelation time and temperature by modifying the galactose removal ratio. However there is little information concerning the morphology and rheological properties of the hydrogel under physiological conditions. Differential scanning microcalorimetry (DSmicroC) showed the thermal gelation process to occur over a broad temperature range (5-50 degrees C). The rheological properties of the hydrogels were investigated as a function of concentration, temperature and ionic strength. The final elastic moduli of the hydrogels increased with increases in concentration. Isothermal rheology suggests that the gelation occurred in two distinct stages, which was influenced by the solution media. Scanning electron microscopy (SEM) was used to characterize the morphology of the xyloglucan which were thermally gelled at 37 degrees C. The resultant morphology was strongly dependent on the concentration of the hydrogel. Strong hydrogels were only obtained at 3 wt.% at 37 degrees C, and the morphology characterized by an open 3-dimensional network, comprised of thin membranes. It is proposed that the first stage of the isothermal gelation is the formation and growth of the thin membranes, followed by the formation of a three dimensional network.

Mechanical effects of plant cell wall enzymes on cellulose/xyloglucan composites.

Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose fibril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.

Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy.

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.