Xylan-Degrading Enzymes from the Thermophilic Fungus Humicola Zanuginosa (Griffon and Maublanc) Bunce: Action Pattern of Xylanase and beta-Glucosidase on Xylans, Xylooligomers and Arabinoxylooligomers

The mode of action of xylanase and beta-glucosidase purified from the culture filtrate of Humicola lanuginosa (Griffon and Maublanc) Bunce on the xylan extracted from sugarcane bagasse and on two commercially available larchwood and oat spelt xylans, on xylooligomers and on arabinoxylooligomers was studied. While larchwood and oat spelt xylans were hydrolyzed to the same extent in 24 h, sugarcane bagasse xylan was hydrolyzed to a lesser extent in the same period. It was found that the rate of hydrolysis of xylooligomers by xylanase increased with increase in chain length, while beta-glucosidase acted rather slowly on all the oligomers tested. Xylanase exhibited predominant ''endo'' action on xylooligomers attacking the xylan chain at random while beta-glucosidase had ''exo'' action, releasing one xylose residue at a time. On arabinoxylooligomers, however, xylanase exhibited ''exo'' action. Thus, it appears that the presence of the arabinose substituent has, in some way, rendered the terminal xylose-xylose linkage more susceptible to xylanase action. It was also observed that even after extensive hydrolysis with both the enzymes, substantial amounts of the parent arabinoxylooligomer remained unhydrolyzed together with the accumulation of arabinoxylobiose. It can therefore be concluded that the presence of the arabinose substituent in the xylan chain results in linkages that offer resistance to both xylanase and beta-glucosidase action.

Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass.

As one of the most abundant polysaccharides on Earth, xylan will provide more than a third of the sugars for lignocellulosic biofuel production when using grass or hardwood feedstocks. Xylan is characterized by a linear β(1,4)-linked backbone of xylosyl residues substituted by glucuronic acid, 4-O-methylglucuronic acid or arabinose, depending on plant species and cell types. The biological role of these decorations is unclear, but they have a major influence on the properties of the polysaccharide. Despite the recent isolation of several mutants with reduced backbone, the mechanisms of xylan synthesis and substitution are unclear. We identified two Golgi-localized putative glycosyltransferases, GlucUronic acid substitution of Xylan (GUX)-1 and GUX2 that are required for the addition of both glucuronic acid and 4-O-methylglucuronic acid branches to xylan in Arabidopsis stem cell walls. The gux1 gux2 double mutants show loss of xylan glucuronyltransferase activity and lack almost all detectable xylan substitution. Unexpectedly, they show no change in xylan backbone quantity, indicating that backbone synthesis and substitution can be uncoupled. Although the stems are weakened, the xylem vessels are not collapsed, and the plants grow to normal size. The xylan in these plants shows improved extractability from the cell wall, is composed of a single monosaccharide, and requires fewer enzymes for complete hydrolysis. These findings have implications for our understanding of the synthesis and function of xylan in plants. The results also demonstrate the potential for manipulating and simplifying the structure of xylan to improve the properties of lignocellulose for bioenergy and other uses.

Kinetic study of hydrolysis of xylan and agricultural wastes with hot liquid water

We investigated the kinetics of hot liquid water (HLW) hydrolysis over a 60-min period using a self-designed setup. The reaction was performed within the range 160-220 °C, under reaction conditions of 4.0 MPa, a 1:20 solid:liquid ratio (g/mL), at 500 rpm stirring speed. Xylan was chosen as a model compound for hemicelluloses, and two kinds of agricultural wastes-rice straw and palm shell-were used as typical feedstocks representative of herbaceous and woody biomasses, respectively. The hydrolysis reactions for the three kinds of materials followed a first-order sequential kinetic model, and the hydrolysis activation energies were 65.58 kJ/mol for xylan, 68.76 kJ/mol for rice straw, and 95.19 kJ/mol for palm shell. The activation energies of sugar degradation were 147.21 kJ/mol for xylan, 47.08 kJ/mol for rice straw and 79.74 kJ/mol for palm shell. These differences may be due to differences in the composition and construction of the three kinds of materials. In order to reduce the decomposition of sugars, the hydrolysis time of biomasses such as rice straw and palm shell should be strictly controlled.

Influence of fibrolytic enzymes on the hydrolysis and fermentation of pure cellulose and xylan by mixed ruminal microorganisms in vitro

A series of in vitro studies was, conducted to determine the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixture (1:1 wt/wt) of both. The enzyme product (Liquicell 2500, Specialty Enzymes and Biochemicals, Fresno, CA) was derived from Trichoderma reesei and contained mainly xylanase and cellulase activities. Addition of enzyme (0.5, 2.55 and 5.1 muL/g of DM) in the absence of ruminal fluid increased (P < 0.001) the release of reducing sugars from xylan and the mixture after 20 h of incubation at 20degreesC. Incubations with ruminal fluid showed that enzyme (0.5 and 2.55 muL/g of DM) increased (P < 0.05) the initial (up to 6 h) xylanase, endoglucanase, and beta-D-glucosidase activities in the liquid fraction by an average of 85%. Xylanase and endoglucanase activities in the solid fraction also were increased (P < 0.05) by enzyme addition, indicating an increase in fibrolytic activity due to ruminal microbes. Gas production over 96 h of incubation was determined using a gas pressure measurement technique. Incremental levels of enzyme increased (P < 0.05) the rate of gas production of all substrates, suggesting that fermentation of cellulose and xylan was enzyme-limited. However, adding the enzyme at levels higher than 2.55 muL/g of DM failed to further increase the rate of gas production, indicating that the maximal level of stimulation was already achieved at lower enzyme concentrations. It was concluded that enzymes enhanced the fermentation of cellulose and xylan by a combination of pre- and postincubation effects (i.e., an increase in the release of reducing sugars during the pretreatment phase and an increase in the hydrolytic activity of the liquid and solid fractions of the ruminal fluid), which was reflected in a higher rate of fermentation.

Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses

Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by alpha-(1,2)- and alpha-(1,3)-linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases alpha-(1,3)-linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for alpha-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.

Digestive and physiologic effects of a wheat bran extract, arabino-xylan-oligosaccharide, in breakfast cereal

Objective: We assessed whether a wheat bran extract containing arabino-xylan-oligosaccharide (AXOS) elicited a prebiotic effect and influenced other physiologic parameters when consumed in ready-to-eat cereal at two dose levels. Methods: This double-blind, randomized, controlled, crossover trial evaluated the effects of consuming AXOS at 0 (control), 2.2, or 4.8 g/d as part of ready-to-eat cereal for 3 wk in 55 healthy men and women. Fecal microbial levels, postprandial serum ferulic acid concentrations, and other physiologic parameters were assessed at the beginning and end of each condition. Results: The median bifidobacteria content of stool samples (log10/grams of dry weight [DW]) was found to be higher in the subjects consuming the 4.8-g/d dose (10.03) than in those consuming 2.2 g/d (9.93) and control (9.84, P < 0.001). No significant changes in the populations of other fecal microbes were observed, indicating a selective increase in fecal bifidobacteria. Postprandial ferulic acid was measured at 120 min at the start and end of each 3-wk treatment period in subjects at least 50 y old (n = 37) and increased in a dose-dependent manner (end-of-treatment values 0.007, 0.050, and 0.069 μg/mL for the control, AXOS 2.2 g/d, and AXOS 4.8 g/d conditions, respectively, P for trend < 0.001). Conclusion: These results indicate that AXOS has prebiotic properties, selectively increasing fecal bifidobacteria, and increases postprandial ferulic acid concentrations in a dose-dependent manner in healthy men and women.

Xylan from corn cobs, a promising polymer for drug delivery: Production and characterization

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of the Lipophilic External Phase Composition on the Preparation and Characterization of Xylan Microcapsules-A Technical Note

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Producing xylan/Eudragit (R) S100-based microparticles by chemical and physico-mechanical approaches as carriers for 5-aminosalicylic acid

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of alpha-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.

A novel xylan degrading beta-D-xylosidase: purification and biochemical characterization

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular beta-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. beta-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 A degrees C and 3.0-5.5, respectively. beta-xylosidase was stable in acidic pH (3.0-6.0) and 70 A degrees C for 1 h. The enzyme was activated by 5 mM MnCl2 (28 %) and MgCl2 (20 %) salts. The beta-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-beta-d-xylopyranoside, exhibiting apparent K-m and V-max values of 0.66 mM and 39 U (mg protein)(-1) respectively, and to a lesser extent p-nitrophenyl-beta-d-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel beta-d-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by beta-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.

Study on the pyrolytic behaviour of xylan-based hemicellulose using TG-FTIR and Py-GC-FTIR

Two sets of experiments, categorized as TG–FTIR and Py–GC–FTIR, are employed to investigate the mechanism of the hemicellulose pyrolysis and the formation of main gaseous and bio-oil products. The “sharp mass loss stage” and the corresponding evolution of the volatile products are examined by the TG–FTIR graphs at the heating rate of 3–80 K/min. A pyrolysis unit, composed of fluidized bed reactor, carbon filter, vapour condensing system and gas storage, is employed to investigate the products of the hemicellulose pyrolysis under different temperatures (400–690 °C) at the feeding flow rate of 600 l/h. The effects of temperature on the condensable products are examined thoroughly. The possible routes for the formation of the products are systematically proposed from the primary decomposition of the three types of unit (xylan, O-acetylxylan and 4-O-methylglucuronic acid) and the secondary reactions of the fragments. It is found that the formation of CO is enhanced with elevated temperature, while slight change is observed for the yield of CO2 which is the predominant products in the gaseous mixture.

Effects of pH on pretreatment of sugarcane bagasse using aqueous imidazolium ionic liquids

Pretreatments of sugarcane bagasse for saccharification using different acid-catalysed imidazolium IL solutions (containing 20% water) at 130 °C for 30 min were investigated. At the same solution pH, pretreatment effectiveness in terms of glucan digestibility, delignification and xylan removal were similar for aqueous 1-butyl-3-methylimidazolium methane sulfonate (BMIMCH3SO3), 1-butyl-3-methylimidazolium methyl sulfate (BMIMCH3SO4), 1-ethyl-3-methylimidazolium chloride (EMIMCl) and 1-butyl-3-methylimidazolium chloride (BMIMCl). Decreasing solution pH of aqueous IL systems from 6.0 to 0.4 increased bagasse delignification and xylan removal, and as a result, improved glucan digestibility. The glucan digestibilities for bagasse samples pretreated by IL solutions with pH ≤ 0.9 were > 90% after 72 h of enzymatic hydrolysis. Without pH adjustment, the effectiveness of these aqueous IL solutions (except BMIMCH3SO3 because of its low natural pH of 0.9) to deconstruct the biomass was poor and the glucan digestibilities of pretreated bagasse samples were < 20%. These results show that pretreatment effectiveness of aqueous imidazolium ILs can simply be estimated from solution pH rather than hydrogen bond basicity (β) of the IL solution.

Low temperature pretreatment of sugarcane bagasse at atmospheric pressure using mixtures of ethylene carbonate and ethylene glycol

A low temperature lignocellulose pretreatment process was developed using acid-catalysed mixtures of alkylene carbonate and alkylene glycol. Pretreatment of sugarcane bagasse with mixtures of ethylene carbonate (EC) and ethylene glycol (EG) was more effective than that with mixtures of propylene carbonate (PC) and propylene glycol (PG). These mixtures were more effective than the individual components in making bagasse cellulose more amenable to cellulase digestion. Glucan digestibilities of ≥87% could be achieved with a wide range of EC to EG ratios from 9:1 to 1:1 (w/w). Pretreatment of bagasse by the EC/EG mixture with a ratio of 4:1 in the presence of 1.2% H2SO4 at 90 °C for 30 min led to the highest glucan enzymatic digestibility of 93%. The high glucan digestibilities obtained under these acidic conditions were due to (a) the ability of alkylene carbonate to cause significant biomass size reduction, (b) the ability of alkylene glycol to cause biomass defibrillation, (c) the ability of alkylene carbonate and alkylene glycol to remove xylan and lignin, and (d) the magnified above attributes in the mixtures of alkylene carbonate and alkylene glycol.

Isolation and Culture of a Thermophilic Fungus, Melanocarpus albomyces, and Factors Influencing the Production and Activity of Xylanase

Summary: An uncommon thermophilic fungus, Melanocarpus albomyces, was isolated from soil and compost by incubating samples in a glucose/sorbose/asparagine liquid medium, followed by enrichment culture in medium containing sugarcane bagasse as carbon source. The culture filtrate protein of the fungus grown in the presence of bagasse or xylose hydrolysed xylan and some other polysaccharides but cellulose was not hydrolysed. High extracellular xylanase (EC 3.2.1.8) activity was produced by cultures grown on xylose or hemicellulosic materials. The enzyme was induced in glucose-grown washed mycelia in response to addition of xylose or xylan but not by alkyl or aryl β-D-xylosides. Cultures produced higher enzyme yields in shaken flasks than in a fermenter. Gel-filtration chromatography of culture filtrate protein showed the presence of two isoenzymes of xylanase, whose relative proportions varied with the carbon source used for growth. The extent of hydrolysis of heteroxylans or the hemicellulosic fraction of bagasse by culture filtrate protein preparations was greater when the cultures had been grown on bagasse rather than xylose as the inducing substrate. The activity of xylanase preparations was increased when an exogenous β-glucosidase was added.