Pure Xylan Research Articles

Isolation of Corncob Xylan-Degrading Fungi and Its Application in Xylobiose Production

In the present study, a potential corncob xylan degradation fungi was isolated and screened from soil to produce xylanase, and was identified as Fusarium oxysporum. The production of xylanase by F. oxysporum under solid state fermentation using corncob powder as the solid substrate reached the maximum xylanase activity when using particle size of substrate of 60 mesh, water content ratio of 2 mL/g substrate, incubation temperature of 30°C, initial pH of 6.0, size of inoculum of 5x107 spore/3 g substrate, and incubation time of 2 days. The xylanase activity increased about 4 times up to 7.92 U/mL after optimization. The potential application of xylanase of F. oxysporum in hydrolyzing alkali-treated corncob xylan to produce xylobiose was also demonstrated. Hydrolysis of 6% of corncob xylan using 100 U/g substrate of enzyme loading under optimum pH and temperature conditions (pH 5.5 and 50°C, respectively) achieved the yield of xylobiose up to 28.7 g/100 g pure xylan after 12 h incubation. The purification of hydrolysate could retain 91.1% of xylobiose. Further separation step using activated charcoal column chromatography was able to get a pure xylobiose, but could only recover 59.3% of xylobiose.

Characterization of a novel Lytic Polysaccharide Monooxygenase from Malbranchea cinnamomea exhibiting dual catalytic behavior

A novel Lytic Polysaccharide Monooxygenase (LPMO) family AA9 (PMO9A_MALCI) protein from thermophilic fungus Malbranchea cinnamomea was cloned and expressed in Pichia pastoris. The expressed protein was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. SDS-PAGE analysis showed PMO9A_MALCI to be ~27 kDa protein. High performance anion exchange chromatography and mass spectrometry confirmed that purified protein was active against an array of cellulosic (avicel, carboxy methyl cellulose) and hemicellulosic (birch wood xylan, wheat arabinoxylan and rye arabinoxylan) substrates, releasing both oxidized and unoxidized cello-oligosaccharide and xylo-oligosaccharide products respectively. Presence of double oxidized products during mass spectrometric analysis as well as in-silico analysis confirmed that the expressed protein belongs to Type 3 LPMO family. Molecular dynamic simulations further confirmed the sharing of common amino acid residues conserved for catalysis of both cellulosic and hemicellulosic substrates which further indicates that both substrates are equally preferred. Enzymatic cocktails constituted by replacing a part of commercial cellulase CellicCTec2 with PMO9A_MALCI (9:1/8:2) led to synergistic improvement in saccharification of acid and alkali pretreated biomass. This is the first report on heterologous expression of LPMO from M. cinnamomea, exhibiting catalysis of cellulose and pure xylan.

Isolation of xylans from bleached Eucalyptus kraft pulp by antisolvents precipitation

The present work is focused on alkaline extraction of xylans from bleached Kraft pulp of Eucalyptus globulus, and its isolation from the alkaline extract, aiming high purity product and yields. Alkaline extraction was optimized, through the stirring speed and extraction time. A higher stirring speed increased extraction yields which varied between 65 and 85%. However, extraction time did not have influence on extraction yields. Xylans isolation was performed by precipitation with diluted acids and with various alcohol concentrations. Washing of the precipitates, to remove impurities, was performed either with water and/or with methanol. Water washing yielded xylans with purity values up to 99%, while with methanol those values were lower, in the range of 36–84%. Xylans characterization indicated average molecular weights of 16.8–22.7 kDa. The isolated xylan yields varied between 43 and 97%. Nitric acid showed to be the most adequate solvent to get pure xylan. On the other hand, in alcohol experiments lower yields were obtained due to the losses in the subsequent water washing process.

Production of prebiotic-xylooligosaccharides from alkali pretreated mahogany and mango wood sawdust by using purified xylanase of Clostridium strain BOH3

Xylooligosaccharides (XOS) are emerging prebiotics which can be produced from lignocellulosic biomass including agro-residues and hardwood. In this study, we report the production of XOS from thermal-alkali pretreated hardwood such as mahogany and mango by using a purified xylanase from Clostridium strain BOH3. In the first approach, pure xylan is extracted from mahogany and mango hardwood and then the pure xylan is hydrolyzed by using the xylanase. In this case, 572 and 504mg XOS/g pure xylan were obtained from mahogany and mango woods, respectively. In the second approach, the same xylanase is employed to hydrolyze sawdust of hardwood after different types of pretreatments. After a thermal (121°C for 15min) pretreatment under a mild alkaline (0.05N NaOH) condition, the pretreated mahogany and mango sawdust can be utilized directly to produce 89.5 and 67.6mg XOS/g pretreated sawdust, respectively. XOS produced from the pretreated sawdust show strong prebiotic effects on Bifidobacteria and Lactobacilli. This report shows the possibility of producing XOS from pretreated woody wastes without using pure xylan as a substrate.

Two degradation strategies for overcoming the recalcitrance of natural lignocellulosic xylan by polysaccharides-binding GH10 and GH11 xylanases of filamentous fungi.

The recalcitrance of lignocellulose forms a strong barrier for the bioconversion of lignocellulosic biomass in chemical or biofuel industries. Filamentous fungi are major plant biomass decomposer, and capable of forming all the required enzymes. Here, they characterized the GH10 and GH11 endo-xylanases and a CE1 acetyl-xylan esterase (Axe1) from a superior biomass-degrading strain, Aspergillus fumigatus Z5, and examined how they interact in xylan degradation. Cellulose-binding (CBM1) domain inhibited GH10 xylanase activities for pure xylan, but afforded them an ability to hydrolyze washed corncob particles (WCCP). CBM1-containing GH10 xylanases also showed synergism with CBM1-containing Axe1 in WCCP hydrolysis, and this synergy was strictly dependent on the presence of their CBM1 domains. In contrast, GH11 xylanases had no CBM1, but still could bind xylan and hydrolyzed WCCP; however, no synergism displayed with Axe1. GH10 xylanases and GH11 xylanases showed a pronounced synergism in WCCP hydrolysis, which was dependent on the presence of the CBM1 in GH10 xylanases and absence from GH11 xylanases. They exhibit different mechanisms to bind to cellulose and xylan, and act in synergy when these two structures are intact. These findings will be helpful for the further development of highly efficient enzyme mixtures for lignocellulosic biomass conversion.

Influence of the biomass components on the pore formation of activated carbon

Uncontrolled management of agricultural wastes have strongly contributed to the increase of greenhouse emissions and pollution. On the other hand, these residues can be used as a sustainable source for the production of activated carbon. Currently, biomasses rich in lignin are the most widely used, due to the high yields and large surface areas attainable. The aim of this study is to understand the influence of each biomass component on activated carbon properties. Alpha-cellulose, xylan, kraft lignin, and mixtures with different ratios of the single components were used as model substances to represent biomass. These materials were pyrolyzed and subsequently activated with KOH to expand the surface area. TGA results showed no interaction between components during pyrolysis but there was a strong influence of the composition of the mixture on the activated carbon properties due to the different thermal stabilities of each char. The activated carbon with the largest apparent surface area was obtained from cellulose with 2220 m2 g−1 and pure xylan showed the lowest with 1950 m2 g−1. T-plot calculations showed that more than 90% of the surface area was composed by micropores. To understand the microporosity, CO2 isotherms were measured. The surface areas calculated were lower but followed the same trend as those obtained from the isotherms with N2.

Synergism of an auxiliary activity 9 (AA9) from Chaetomium globosum with xylanase on the hydrolysis of xylan and lignocellulose

Auxiliary activity family 9 (AA9) is a group of fungal synergistic proteins belonging to the lytic polysaccharide monooxygenases. Via oxidative cleavage, AA9 facilitates cellulose hydrolysis by cellulase. However, oxidative activity of AA9 on pure xylan has not been reported yet. Previously, an AA9 from Chaetomium globosum (CgAA9) was identified and expressed in Escherichia coli. The recombinant CgAA9 exhibited synergistic effects in cellulose hydrolysis. Here, we showed that CgAA9 acts synergistically in xylan hydrolysis. When the insoluble fraction of xylan from beechwood was hydrolyzed by xylanase, CgAA9 yielded 1.3 times more reducing sugar than that produced only by xylanase, and the extent of synergism by CgAA9 was enhanced in the presence of ascorbic acid as a reducing cofactor. In addition, CgAA9 functioned on pretreated rice straws. This is the first report of the synergistic activity of AA9 with xylanase in the hydrolysis of pure xylan and xylan contained in pretreated lignocellulose.

Effect of salicin on induction and carbon catabolite repression of endoxylanase synthesis in Penicillium janthinellum MTCC 10889

AbstractAmongst various carbon sources, xylan was found to be the sole inducer of endoxylanase production by Penicillium janthinellum MTCC 10889 in submerged cultivation. Endoxylanase synthesis by a xylan induced culture was initially repressed after a simultaneous addition of xylose, probably by the inducer exclusion mechanism, but it was resumed and achieved its highest level at a much later stage of growth (at 120 h). Xylose added after 30 h of growth cannot exert its full repressive effect. Although glucose was proved to be a more potent repressor than xylose, supplementation of salicin, an alcoholic β-glycoside containing d-glucose, with pure xylan resulted in an about 3.22 fold increase in the enzyme synthesis at 72 h followed by constant high production of the enzyme at least until the 144th h of growth. Inducing capacity of salicin in a xylan induced culture was significantly reduced when it was added after 30 h of growth. Addition of salicin and xylan help to partially overcome the repressive effect of xylose and glucose. Failure of salicin in recovering the endoxylanase synthesis in actinomycin D and cyclohexamide inhibited the xylan induced culture indicating that salicin cannot initiate the de novo synthesis of the enzyme.

Hydroxyalkylated xylans – Their synthesis and application in coatings for packaging and paper

This paper demonstrates opportunities for wood-based xylan derivatives to be used in coating applications. A route for extraction and purification of white and pure xylan from bleached birch kraft pulp is described as a part of the production of high adsorption and high crystalline pulp. Derivatization of the xylan during the extraction step was also demonstrated. Efficient derivatization of xylan to water soluble derivatives was achieved and promising results were obtained in primary application tests as a coating component in barrier coatings on board and as a binder component in pigment coating of offset paper. With the best xylan derivate coating, the barrier properties were better than with a commercial biopolymer coating, while oxygen permeability was roughly one third of that for a polyethylene terephthalate coating. Likewise, surface strength close to the reference latex as a binder in pigment coatings was achieved by a xylan derivative. This work is part of a platform of hemicellulose derivatives enabling novel application for this medium to high molar mass hemicellulose of high purity.

Produciton of xylo-oligosaccharides by chemo-enzymatic treatment of agricultural by-products

Several timbers and crop by-products were subjected to an enzymatic treatment to obtain a xylo-oligosaccharides-enriched preparation. The process was performed by means of the commercial endo-β-1,4-xylanase Buzyme 2511®. The enzymatic cocktail was applied onto the raw ground materials with yield up to 5.3 ± 1.0 g/L xylo-oligosaccharides for apple pomace. In order to make the materials more accessible to enzymatic hydrolysis, they were subjected to thermal-alkaline treatment. The biocatalysis process over the thermo-alkaline treated materials yielded xylo-oligosaccharide solutions with the following concentrations (g/l): 1.3 white poplar (Populus alba), 2.9 giant cane (Arundo donax), 3.7 apple pomace (Malus domestica), and 6.5 stalk of grapes (Vitis vinifera). The preparation resulting from biotransformation of grape stalk contained mostly xylo-oligosaccharides (96% w/v) with a small amount of xylose (3% w/v). The same ratio was obtained when pure xylan from birchwood was used as feedstock.

Application of cellulase and hemicellulase to pure xylan, pure cellulose, and switchgrass solids from leading pretreatments

Accellerase 1000 cellulase, Spezyme CP cellulase, β-glucosidase, Multifect xylanase, and beta-xylosidase were evaluated for hydrolysis of pure cellulose, pure xylan, and switchgrass solids from leading pretreatments of dilute sulfuric acid, sulfur dioxide, liquid hot water, lime, soaking in aqueous ammonia, and ammonia fiber expansion. Distinctive sugar release patterns were observed from Avicel, phosphoric acid swollen cellulose (PASC), xylan, and pretreated switchgrass solids, with accumulation of significant amounts of xylooligomers during xylan hydrolysis. The strong inhibition of cellulose hydrolysis by xylooligomers could be partially attributed to the negative impact of xylooligomers on cellulase adsorption. The digestibility of pretreated switchgrass varied with pretreatment but could not be consistently correlated to xylan, lignin, or acetyl removal. Initial hydrolysis rates did correlate well with cellulase adsorption capacities for all pretreatments except lime, but more investigation is needed to relate this behavior to physical and compositional properties of pretreated switchgrass.

Fast enzymatic saccharification of switchgrass after pretreatment with ionic liquids

The pretreatment of cellulose using ionic liquids (ILs) has been shown to be an effective method for improving the enzymatic hydrolysis of cellulose; this technique affords a fast and complete saccharification of cellulose into reducing sugars (Dadi et al., Biotechnol Bioeng. 2006; 95:904-910; Liu and Chen, Chinese Sci Bull. 2006; 51:2432-2436; Zhao et al., J Biotechnol. 2009; 139:47-54). Motivated by these advances, this study examines the effect of IL-pretreatment on the enzymatic hydrolysis of purified xylan (as a model system of hemicellulose) and switchgrass (as a real lignocellulose). The IL-pretreatment resulted in no improvement in the hydrolysis of xylan. The likely reason is that pure xylan has a low degree of polymerization (DP), and is readily biodegraded even without any pretreatment. However, in real cellulosic materials (such as switchgrass), xylan is entrapped within the cellulosic matrix, and cannot be conveniently accessed by enzymes. Our data demonstrate that the IL-pretreatment of switchgrass significantly improved the enzymatic saccharification of both cellulose (96% D-glucose yield in 24 h) and xylan (63% D-xylose yield in 24 h). The compositional analysis of switchgrass suggests a lower lignin content after IL-pretreatment. In addition, the infrared spectrum of regenerated switchgrass indicates a lower substrate crystallinity, whereas the enzyme adsorption isotherm further implies that the regenerated substrate is more accessible to enzymes. This study has further confirmed that IL-pretreatment is an effective tool in enhancing the enzymatic hydrolysis of cellulosic biomass, and allowing a more complete saccharification.

Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies

Moderate loadings of cellulase enzyme supplemented with beta-glucosidase were applied to solids produced by ammonia fiber expansion (AFEX), ammonia recycle (ARP), controlled pH, dilute sulfuric acid, lime, and sulfur dioxide pretreatments to better understand factors that control glucose and xylose release following 24, 48, and 72 h of hydrolysis and define promising routes to reducing enzyme demands. Glucose removal was higher from all pretreatments than from Avicel cellulose at lower enzyme loadings, but sugar release was a bit lower for solids prepared by dilute sulfuric acid in the Sunds system and by controlled pH pretreatment than from Avicel at higher protein loadings. Inhibition by cellobiose was observed to depend on the type of substrate and pretreatment and hydrolysis times, with a corresponding impact of beta-glucosidase supplementation. Furthermore, for the first time, xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, and xylose, xylobiose, and xylotriose were shown to have progressively greater effects on hydrolysis rates. Consistent with this, addition of xylanase and beta-xylosidase improved performance significantly. For a combined mass loading of cellulase and beta-glucosidase of 16.1 mg/g original glucan (about 7.5 FPU/g), glucose release from pretreated solids ranged from 50% to75% of the theoretical maximum and was greater for all pretreatments at all protein loadings compared to pure Avicel cellulose except for solids from controlled pH pretreatment and from dilute acid pretreatment by the Sunds pilot unit. The fraction of xylose released from pretreated solids was always less than for glucose, with the upper limit being about 60% of the maximum for ARP and the Sunds dilute acid pretreatments at a very high protein mass loading of 116 mg/g glucan (about 60 FPU).

Characterization of the degree of polymerization of xylooligomers produced by flowthrough hydrolysis of pure xylan and corn stover with water

Mechanisms that control xylan removal during pretreatment of lignocellulosic biomass are not well understood. For example, although hemicellulose hydrolysis is virtually always assumed to follow first-order homogeneous kinetics, the increase in xylan removal with flow rate for flowthrough pretreatment systems is inconsistent with the predictions for such models, and better information is needed to understand the causes of such discrepancies. Thus, new methods were developed to follow the fate of xylooligomers with degrees of polymerization of up to 30, a range not possible before, for water-only flowthrough pretreatment of oat spelt xylan and corn stover for temperatures of 200–240 °C. Material balances based on the oligomer release profiles produced by batch and flowthrough operations could be closed, suggesting the methods were quite accurate. However, the results also showed that increasing the flow rate from 0 to 2 and then 25 mL/min affected the size distribution of the xylan oligomers (DP < 30) released from corn stover but not from oat spelt xylan and also increased overall hemicellulose sugar solubilization. One explanation for these difference is that lignin and lignin–xylan compounds in particular play an important role in the hydrolysis of lignocellulosic biomass.

Production and Characterization of Films from Cotton Stalk Xylan

Composite film production based on cotton stalk xylan was studied, and the mechanical and physical properties of the films formed were investigated. Xylan and lignin were separated from cellulose by alkali extraction and, then, lignin was removed using ethanol washing. Self-supporting continuous films could not be produced using pure cotton stalk xylan. However, film formation was achieved using 8-14% (w/w) xylan without complete removal of lignin during xylan isolation. Keeping about 1% lignin in xylan (w/w) was determined to be sufficient for film formation. Films were produced by casting the film-forming solutions, followed by solvent evaporation in a temperature (20 degrees C) and relative humidity (40%) controlled environment. The elastic modulus and hypothetical coating strength of the films obtained by using 8% xylan were significantly different from the ones containing 10-14% xylan. The water vapor transfer rates (WVTR) decreased with increasing xylan concentration, which made the films thicker. The glycerol addition as an additional plasticizer resulting in more stretchable films having higher WVTR and lower water solubility values. As a result, film production was successfully achieved from xylan, which was extracted from an agricultural waste (cotton stalk), and the film-forming effect of lignin on pure xylan has been demonstrated.

Xylan/polyvinyl alcohol blend and its performance as hydrogel

AbstractXylan was first modified with maleic anhydride (MA) to obtain xylan‐MA. The derivative was then blended with polyvinyl alcohol (PVA) to generate xylan‐MA/PVA hydrogel. Xylan‐MA was prepared by reacting xylan with MA under an acidic condition. A ring opening of MA occurred, yielding ester linkages between xylan and MA, as verified by 13C‐NMR and FTIR. The effects of reaction temperature and weight ratio of xylan to MA on esterification were examined. Thermal stability of modified xylan derivatives was compared with that of pure xylan using TGA. The blends of xylan‐MA with PVA became crosslinked gel after being heated at high temperature. The influences of MA and PVA contents on strength and swelling behaviors of the gels were investigated. The cytotoxicity of the gels was also evaluated. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 1914–1918, 2006

Synthesis and regulation of D-xylanase from Cellulomonas flavigena wild type and a mutant

The xylanolytic system from Cellulomonas flavigena was enhanced by adding cellulose to the growth medium. The Solka floc:xylan (60:40 w/w) mixture induced xylanase synthesis by more than 3-fold over that induced by growing C. flavigena, wild type and its mutant PN-120 on pure xylan. The hydrolysis pattern of sugar cane bagasse and xylan indicated the presence of debranching endo-β;-xylanase activity.

Separation, characterization and hydrogel-formation of hemicellulose from aspen wood

Hemicellulose from aspen ( Populus tremula) was isolated by an alkali extraction method, which was followed by hydrogen peroxide treatment, ultrafiltration and recovery by spray drying. The sugar composition and lignin content were monitored with HPLC at each step of the separation procedure. Size-exclusion chromatography showed a polymeric hemicellulose of relatively high molar mass. The product was characterized by 1H and 13C NMR spectroscopy and was found to be composed of a linear (1→4)-β-linked d-xylose main chain with a 4- O-methyl-α- d-glucuronic acid substituting the 2-position of approximately every eighth xylose unit. Lignin and O-acetyl groups had largely been removed in the separation process. The xylan was soluble in hot water, and the film forming properties were examined at various mixtures of the hemicellulose and chitosan. These films formed hydrogels with a high swelling capacity at certain compositions. The morphologies of the films were examined with wide angle X-ray spectroscopy, and a pure xylan film was found to be crystalline, which was suggested to be a consequence of the lack of O-acetyl groups. The crystallinity of the films was found to decrease with an increasing amount of chitosan, and the film of chitosan alone showed no crystallinity. The cohesive forces of the hydrogels are suggested to be the result of the crystalline arrangement of the polymers and of electrostatic interactions between acidic groups in the hemicellulose and amine groups in the chitosan.

Effect of the temperature of extraction on the composition of cell wall polysaccharides in olives

A comparative study of methods for isolating cell wall material from olives (variety Hojiblanca) was performed. Treatment with cold (T-1) or hot (T-2) ethyl alcohol was followed by extraction into cold or hot water (water-soluble fraction). The insoluble residues were delignified. Hemicelluloses A and B were isolated and the neutral sugar composition and protein content of the different fractions were determined. In the delignified fractions following treatments T-1 and T-2, hemicelluloses A were composed of 87.35% and 87.37% xylose, respectively, indicating the presence of a pure xylan. In the non-delignified fraction, the content of hemicelluloses B was higher than in the delignified fraction and its predominant sugar was glucose, although significant amounts of xylose, galactose and arabinose were also found. The study suggests that treatment T-1 gives better results than T-2 and that a higher degree of purification of hemicelluloses A is obtained from the delignified cell wall material, as well as a decrease in the proteins bound to the fractions.

Hemicellulolytic enzymes in P- and S-strains of Heterobasidion annosum

Summary: The secretion of several hemicellulolytic enzymes and endoglucanase was studied in S- and P-group strains of a wood-rotting pathogen, Heterobasidio annosum, using enzyme activity assays and isoelectric focusing. In liquid cultures supplemented with birch sawdust or pure xylan, both strains of the fungus produced xylanases, mannanases, endoglucanases and α-galactosidases. Low activities were detected in cultures containing only glucose as a carbon source. When S-strain cultures were provided with a cart matrix, protein secretion to the culture medium started earlier and was significantly increased compared to free cell cultures. This was not the case with the P-strain. In both strains of H. annosum the pH of the birch sawdust culture medium fell within 12 d to below 3.5, and then started to increase towards the end of the cultivation period. The increase in pH appeared to corresprelate with the formation of hemicellulolytic and endoglucanase activitie. The isoenzyme patterns of xylanase, mannanase and endoglucanase were typical for P- and S-strains. In P-strain cultures containing xylan, a xylanase with an alkaline pl value was induced which, however, was absent when the strain was grown with the birch sawdust substrate. In the P-strain the mannanase pattern at the early stage of growth was comparable to that of t S-strain at the later stages of growth on birch sawdust medium. Later in the growth of the P-strain, this pattern was replaced by mannanases with more acid pl values. The relative intensities of the individual endoglucanases varie between the P- and S-strains.