Water-unextractable Arabinoxylan Research Articles

Kinetics and substrate selectivity of a Triticum aestivum xylanase inhibitor (TAXI) resistant D11F/R122D variant of Bacillus subtilis XynA xylanase

This study examined the kinetics and substrate selectivity of a GH11 Bacillus subtilis XynA xylanase (BsX) sensitive to inhibition by TAXI and an engineered variant, which is much less inhibited by TAXI (BsX mut). The main purpose of the work was to elucidate any influence of the structural point mutations on the kinetics and substrate selectivity of the enzyme. Three-dimensional structures of both xylanases were superimposed to elucidate the structural basis for differences in their hydrolytic properties. The two xylanases were incubated individually with water-extractable arabinoxylan (WEAX), water-unextractable arabinoxylan (WUAX), birchwood xylan, and wheat bran. Both the BsX and the BsX mut catalyzed the release of xylo-oligosaccharides with higher degree of polymerization from WUAX than from WEAX. At equimolar addition levels the activity of the BsX mut was lower than that of the BsX with respect to both the initial rate and the product yields obtained after prolonged reaction on the xylan substrates. The calculated substrate selectivity factors indicated that the BsX and the BsX mut both had higher catalytic rate on WUAX than on WEAX. Addition of a 100:1 (TAXI:xylanase) molar ratio of inhibitor confirmed the significantly decreased inhibition of BsX mut by TAXI. Addition of TAXI also influenced the xylanases’ selectivity factor differently.

Evaluation of the prebiotic properties of wheat arabinoxylan fractions and induction of hydrolase activity in gut microflora

Dietary supplementation with prebiotics may result in the stimulation of the growth of beneficial bacteria such as lactobacilli and bifidobacteria in the human gastrointestinal tract. The effect of water-unextractable arabinoxylans (WU-AX) derived from wheat on the modulation of gut bacterial composition was investigated using a mixed culture fermentation system. A prebiotic index (PI) score of 2.03 was obtained after addition of 1% (w/v) WU-AX to a pH-controlled stirred anaerobic fermentation vessel. Pretreatment of the WU-AX with endo-β-1,4-xylanase resulted in significantly higher PI value (3.48) indicating that pretreatment provided oligomers that were better utilised by the gut bacteria. The extracellular hydrolytic enzymes xylanase and ferulic acid esterase are both required for bacterial metabolism of WU-AX and both activities were present in supernatants derived from the mixed batch cultures. Addition of the WU-AX substrates to the batch cultures produced several fold increases of bacterial synthesis of both enzymes, and these increases were greater when the WU-AX substrate was pretreated with xylanase.

In vitro three-stage continuous fermentation of wheat arabinoxylan fractions and induction of hydrolase activity by the gut microflora

In vitro fermentations were carried out by using a model of the human colon to stimulate microbial activities of gut bacteria. The model consisted of a three-stage culture system. Bacterial populations were evaluated under the effect of three types of arabinoxylan, a nonstarch polysaccharide derived from wheat, the water-unextractable arabinoxylan fraction (WU-AX), WU-AX pretreated with exogenous xylanase and the soluble water-extractable arabinoxylan fraction (WE-AX). The xylanase pretreated (WU-AX) had a stimulatory effect upon colonic bifidobacteria throughout all three vessels. Counts of Bacteroides spp. and Clostridium spp. were also both significantly reduced. Addition of the WU-AX substrates to the first vessel resulted in induction of bacterial synthesis of extracellular hydrolytic enzymes xylanase and ferulic acid esterase which are both required for bacterial metabolism of WU-AX; this induction was significantly greater with the xylanase treated WU-AX.

Alteration of Bacillus subtilis XynA endoxylanase substrate selectivity by site-directed mutagenesis

Bacillus subtilis endoxylanase XynA was engineered by site-directed mutagenesis to alter its substrate selectivity, i.e. the ratio of its capacity to solubilise water-unextractable arabinoxylans (WU-AX) to its capacity to hydrolyse water-extractable AX (WE-AX). Four surface exposed aromatic residues were targeted for mutation to alanine, either individually (Y113 and W185) or jointly (F48, Y94, Y113 and W185), based on the general role of aromatic residues in the binding of endoxylanases to insoluble substrates and the hypothesis that they affect the activity of endoxylanases towards WU-AX. Determination of the substrate selectivity factors and WU-AX degradation profile of the wild type and mutant enzymes using an elaborate procedure requiring incubation of native wheat flour WU-AX and WE-AX with different enzyme levels showed that mutants W185A and 4mutA, the mutant containing all four mutations, displayed lower selectivity towards WU-AX than the wild type recombinant endoxylanase. Each of the endoxylanases had similar binding activity to wheat flour WU-AX and insoluble oat spelt xylan.

Effects of cell wall components on the functionality of wheat gluten

Normal white wheat flours and especially whole meal flour contain solids from the inner endosperm cell walls, from germ, aleurone layer and the outer layers of cereal grains. These solids can prevent either gluten formation or gas cell structure. The addition of small amounts of pericarp layers (1–2%) to wheat flour had a marked detrimental effect on loaf volume. Microstructural studies indicated that in particular the epicarp hairs appeared to disturb the gas cell structure. The detrimental effects of insoluble cell walls can be prevented by using endoxylanases. It has been shown that some oxidative enzymes, naturally present in flour or added to the dough, will oxidise water-extractable arabinoxylans via ferulic acid bridges, and the resulting arabinoxylan gel will hinder gluten formation. The negative effects of water-unextractable arabinoxylans on gluten yield and rheological properties can be compensated by the addition of ferulic acid. Free ferulic acid can probably prevent arabinoxylan cross-linking via ferulic acid.

Effect of the proteinaceous wheat xylanase inhibitor XIP‐I on the performances of an Aspergillus niger xylanase in breadmaking

AbstractThe functionality of the proteinaceous wheat xylanase inhibitor XIP‐I in Aspergillus niger endoxylanase‐aided breadmaking was investigated using small‐scale breadmaking and in vitro experiments. At an optimal dosage for an increase in bread volume, the endoxylanase provoked a ∼15% increase of loaf volume in standard flour experiments, but the dough was significantly more sticky than in the control. No variations in bread volumes were observed in the presence of added XIP‐I in endoxylanase dough but stickiness was much reduced. Arabinoxylan (AX) solubilisation from flour cell wall material was practically unaffected with XIP‐I, and the viscosity of dough water extracts and AX intrinsic viscosity were slightly enhanced. In other breadmaking experiments using a flour from which water extractable material was inactivated, or a reconstituted flour (starch, gluten, water extractable arabinoxylans (WEAXs), water unextractable arabinoxylans (WUAXs)), no differences in loaf volumes were observed between control, endoxylanase and endoxylanase/XIP‐I breads, but the high stickiness induced by endoxylanase was totally corrected by XIP‐I. In these doughs, AX solubilisation was slightly affected by XIP‐I whereas water extract viscosity and WEAX intrinsic viscosity were much higher than in endoxylanase doughs. In vitro experiments with purified AX and A. niger endoxylanase and XIP‐I confirmed these findings, showing that the endoxylanase inhibition by XIP‐I had more effect on the characteristics measured on WEAX (specific and intrinsic viscosities) than those measured on WUAX (AX solubilisation). Premixing substrate, endoxylanase and inhibitor in different combinations showed that XIP‐I had a tendancy to bind WUAX, but not WEAX, which changed the balance between enzymic AX solubilisation and AX depolymerisation. As a result, a high viscosity of water extracts was preserved, which is beneficial for dough handling properties. Copyright © 2006 Society of Chemical Industry

Evidence for the Involvement of Arabinoxylan and Xylanases in Refrigerated Dough Syruping

The relationship between syruping in refrigerated doughs upon prolonged storage and different aspects of arabinoxylan (AX) hydrolysis was investigated using Triticum aestivum xylanase inhibitor (TAXI) and different xylanases in the dough formula. Dough characteristics were evaluated with strong emphasis on the AX population and its fate as a function of storage time. Selective reduction of part of the flour endogenous xylanase activity in dough by added TAXI reduced dough syruping after 12 and 20 days of storage by 50%, providing straightforward evidence for the involvement of xylanases and, thus, AX in the syruping phenomenon. Addition of xylanases with different inhibitor sensitivities [an inhibition-sensitive Bacillus subtilis xylanase (XBS(i)) as well as a noninhibited mutant (XBS(ni)) thereof] to dough confirmed the importance of xylanases in dough syruping, on one hand, and the power of wheat flour TAXI to constitute a significant barrier against xylanase-mediated dough syruping, on the other hand. Use of xylanases with different substrate selectivities [an Aspergillus aculeatusxylanase (XAA) versus XBS(ni)] showed degradation of water-extractable AX (WE-AX) and solubilized AX to low molecular weight molecules rather than the conversion of water-unextractable AX (WU-AX) to high molecular weight water extractable components to be the main factor influencing dough syruping.

Enzymic Degradability of Hull-less Barley Flour Alkali-Solubilized Arabinoxylan Fractions by Endoxylanases

The impacts of the arabinose to xylose (A/X) ratio of arabinoxylans (AX) and the endoxylanase substrate specificity on the enzymic degradability of hull-less barley flour AX by endoxylanases were studied by using alkali-solubilized AX (AS-AX) fractions with different A/X ratio, on the one hand, and glycoside hydrolase family 10 and 11 endoxylanases of Aspergillus aculeatus (XAA) and Bacillus subtilis (XBS), respectively, on the other hand. AS-AX were obtained by saturated barium hydroxide treatment of hull-less barley flour water-unextractable AX. Fractionation of AS-AX by stepwise ethanol precipitation resulted in structurally different hull-less barley flour AS-AX fractions. Their A/X ratios increased with increasing ethanol concentration, and this increase in A/X ratio was reflected in their xylose substitution levels. For both XAA and XBS, the enzymic degradability of AX and apparent specific endoxylanase activity decreased with increasing A/X ratio of the AS-AX substrates, implying that both endoxylanases were sterically hindered by arabinose substituents. However, for all AS-AX fractions, hydrolysis end products of lower average degree of polymerization were obtained after incubation with XAA than with XBS, indicating that the former enzyme has a lower substrate specificity toward hull-less barley flour AS-AX than the latter. In addition, apparent specific endoxylanase activities indicated that XBS was approximately 2 times more sensitive to variations in the A/X ratio of AS-AX fractions than XAA. Furthermore, AS-AX with higher A/X ratio were relatively resistant to degradation by XBS.

Endoxylanase substrate selectivity determines degradation of wheat water-extractable and water-unextractable arabinoxylan

The relative activity of an endoxylanase towards water-unextractable (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, referred to as endoxylanase substrate selectivity, impacts the enzyme functionality in cereal-based biotechnological processes such as bread-making and gluten starch separation. A set of six endoxylanases representing a range of substrate selectivities as determined by a screening method using chromophoric substrates [ Anal. Biochem. 2003, 319, 73–77] was used to examine the impact of such selectivity on changes in structural characteristics of wheat WU-AX and WE-AX upon enzymic hydrolysis. While WE-AX degradation by the selected endoxylanases was very comparable with respect to apparent molecular mass (MM) profiles and arabinose to xylose ratio of the hydrolysates formed, WU-AX solubilisation and subsequent degradation of solubilised fragments gave rise to widely varying MM profiles, depending on the substrate selectivity of the enzymes. Enzymes with high selectivity towards WU-AX de facto generated higher MM fragments from WU-AX than enzymes with low selectivity. The arabinose to xylose ratios of solubilised fragments were independent of the degree of solubilisation.

The bread-making functionalities of two Aspergillus niger endoxylanases are strongly dictated by their inhibitor sensitivities

A recent approach based on affinity chromatography with immobilised endoxylanase inhibitors was used to isolate two endoxylanases (EC 3.2.1.8) with different bread-making functionalities from an Aspergillus niger fermentation broth. TAXI ( Triticum aestivum endoxylanase inhibitor) affinity chromatography yielded a TAXI- and XIP (endoxylanase inhibiting protein)-sensitive family 11 endoxylanase (24 kDa, p I 3.5), XIP affinity chromatography subsequently yielded a family 10 endoxylanase (36 kDa), only inhibited by XIP. While the first enzyme improves bread volume, the latter enzyme has no effect on bread quality whatsoever. The bread-making positive endoxylanase rather selectively hydrolyses water-unextractable arabinoxylan in an in vitro screening method, still performs/is active during bread-making and produces soluble arabinoxylan of high (>11.2 × 10 4 Da) and low molecular mass (≤11.2 × 10 4 Da). In contrast, the bread-making neutral endoxylanase in the in vitro assay displays a bias for water-extractable arabinoxylan and is immediately and almost completely inhibited during the early stages of bread-making. The results show that the functionalities of the purified A. niger endoxylanases in wheat bread-making are strongly dictated by their sensitivities towards wheat endoxylanase inhibitors.

Water-Extractable and Water-Unextractable Arabinoxylans Affect Gluten Agglomeration Behavior during Wheat Flour Gluten−Starch Separation

Water-extractable arabinoxylan (WE-AX) of variable molecular weight (MW) and water-unextractable arabinoxylan (WU-AX) were added to wheat flour to study their effect on gluten agglomeration in a dough and batter gluten-starch separation process with recovery of gluten from the batter with a set of vibrating sieves (400, 250, and 125 microm). Low MW WE-AX had almost no impact on the distribution of the gluten on the different sieves. High MW WE-AX decreased yields of the largest (400 microm sieve) gluten aggregates, more than their medium MW counterparts, indicating the importance of AX MW for their effect on gluten interactions. Correlations between the total level of gluten protein recovered on the three sieves and the batter extract viscosity as well as between the proportion of gluten protein recovered on the 400 microm sieve to that on the three sieves and the batter extract viscosity pointed to the importance of viscosity as an indicator for gluten agglomeration, as did the fact that another viscosity increasing plant polysaccharide (guar gum) also negatively influenced gluten agglomeration. However, the obtained data cannot rule out that AX and guar gum also exert steric effects on gluten agglomeration. WU-AX, present as discrete cell wall fragments, had a negative impact on the level of large gluten aggregates. Taken together, the results show that both native WE-AX and WU-AX detrimentally impact gluten agglomeration.

The combined use of hull-less barley flour and xylanase as a strategy for wheat/hull-less barley flour breads with increased arabinoxylan and (1→3,1→4)-β-D-glucan levels

Bread-making with a composite flour (CF) consisting of 60% wheat flour (WF) and 40% hull-less barley flour, increased the total and soluble (1→3,1→4)-β-D-glucan and total arabinoxylan (AX) contents of dough and bread samples, but decreased the specific bread loaf volume. A xylanase insensitive to inhibition by Triticum aestivum L. xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP), increased loaf volume by 8.8 and 20.1% for WF and CF breads, respectively. Xylanase addition not only markedly improved loaf volume of CF bread, but also increased the soluble AX content of the WF and CF dough and bread samples because of conversion of water-unextractable AX into soluble AX. The xylanase had no impact on the extractability and molecular weight of (1→3,1→4)-β-D-glucan, but (1→3,1→4)-β-D-glucan was degraded during bread-making probably because of endogenous β-glucanase activity. Taken together, the results clearly show that the combined use of hull-less barley flour and a xylanase active during bread making, lead to palatable breads with high total and soluble AX and (1→3,1→4)-β-D-glucan contents. The sum of total AX and (1→3,1→4)-β-D-glucan was 1.70% for WF bread and 3.06% for CF bread, while the sum of soluble AX and (1→3,1→4)-β-D-glucan was 0.49 and 1.41% for control WF and CF xylanase supplemented breads, respectively.

Impact of inhibition sensitivity on endoxylanase functionality in wheat flour breadmaking.

A Bacillus subtilis endoxylanase (XBS(i)) sensitive to inhibition by Triticum aestivum L. endoxylanase inhibitor (TAXI) and a mutant thereof (XBS(ni)), uninhibited by TAXI, were used in straight-dough breadmaking to assess the importance of endoxylanase inhibition sensitivity on endoxylanase functionality in the process. With two European wheat flours, the loaf volume improving effect of XBS(ni) at much lower enzyme dosages was substantially larger than that brought about by XBS(i). This coincided with differences in arabinoxylan (AX) hydrolysis. Although XBS(ni) had a lower substrate selectivity for water-unextractable arabinoxylan (WU-AX) than XBS(i), the former solubilized significantly more WU-AX than XBS(i). Because of inhibition, XBS(i) solubilized most of the WU-AX during mixing, whereas, with XBS(ni), the rate of solubilization decreased less with increasing processing time than that with XBS(i). During fermentation and baking and at the highest dosage (600 U/kg of flour of XBS(i) and 60 U/kg of flour of XBS(ni)), XBS(ni) induced a stronger degradation of enzymically solubilized and water-extractable AX than XBS(i). Taken together, the data clearly demonstrate that endoxylanases, which in vitro are inhibited by endoxylanase inhibitors and still are active in the breadmaking process, as demonstrated by their functional (bread volume) enhancing effect, gradually lose their activity in the process.

Enzymatic solubilization of arabinoxylans from native, extruded, and high-shear-treated rye bran by different endo-xylanases and other hydrolyzing enzymes.

The overall objective of this research was to find a new way to valorize rye bran, by producing a gellifier from the enzymatic solubilization of arabinoxylans (AX). The effects of three pure endo-xylanases from Aspergillus niger (Xyl-1), Talaromyces emersonii (Xyl-2), and Bacillus subtilis (Xyl-3) and of Grindamyl S100 (GS100), a commercial enzyme preparation containing a Xyl-1 type endo-xylanase, were tested on rye bran to study the solubilization of water-unextractable arabinoxylans (WUAX). Eight different extrusion-treated rye brans were also used as substrates to find the best physical treatment to facilitate enzymatic arabinoxylan (AX) solubilization. Arabinoxylans were better solubilized from the bran extruded at high temperature using Xyl-3. This enzyme was then tested in combination with pure (1,4)-beta-d-arabinoxylan arabinofuranohydrolase (AXH) and endo-beta-d-glucanase or ferulic acid esterase (FAE), from A. niger. Only beta-glucanase in combination with Xyl-3 improved the AX extraction, but it did not have a marked effect on the viscosity of the extracts. Xyl-3 was then tested on a high-shear-treated rye bran, and results were compared to those obtained with the high-temperature-extruded rye bran. The high-shear treatment did not improve the bran AX enzymatic solubilization. The combination of FAE with Xyl-1 or Xyl-3 did not improve the AX extraction from untreated and high-shear-treated rye bran. Finally, to study the gelation capacity of the enzymatically solubilized AX, the effect of the hydrogen peroxide/horseradish peroxidase (H(2)O(2)/POD) was tested on the Xyl-3 high-temperature-extruded bran extracts. Solubilized AX did not gel in the presence of the oxidizing system.

Substrate selectivity and inhibitor sensitivity affect xylanase functionality in wheat flour gluten–starch separation

Addition of xylanases (EC 3.2.1.8) that varied in their substrate selectivities and/or wheat xylanase inhibitor sensitivities in dough batter gluten–starch separation of wheat flour showed the importance of these enzyme characteristics for their functionality in this process. A xylanase from Aspergillus aculeatus (XAA) with selectivity for hydrolysis of water extractable arabinoxylan (WE-AX), which is not inhibited by wheat flour xylanase inhibitors decreased batter viscosity and improved gluten agglomeration behaviour. In contrast, a xylanase from Bacillus subtilis (XBSi) with selectivity for hydrolysis of water unextractable arabinoxylan (WU-AX), which is in vitro inhibited by wheat flour xylanase inhibitors had a negative effect on gluten agglomeration at low enzyme dosages. As expected, solubilisation of WU-AX increased batter viscosities. At higher dosages however, this enzyme also improved gluten agglomeration because of degradation of both WE-AX and enzymically solubilised AX. A mutated B. subtilis xylanase (XBSni) with selectivity for hydrolysis of WU-AX comparable to XBSi but which is not inhibited by wheat flour xylanase inhibitors, increased the level of large gluten aggregates as well as the total gluten protein recovery, even at lower dosages. Because of its inhibitor insensitivity, the solubilisation and degradation of AX proceeded further. An XBSni dosage approximately 4 times lower than XBSi performed as well as its inhibited counterpart. The degradation of both WE-AX and WU-AX by XBSni improved the gluten agglomeration behaviour to a larger extent than the XAA treatment which primarily resulted in hydrolysis of WE-AX. The results confirm the detrimental impact not only of WE-AX, but also of WU-AX, on gluten agglomeration in a dough batter gluten–starch separation process. At the same time, they provide firm evidence that xylanases are not only inhibited by xylanase inhibitors in vitro, but are also partly inhibited in the industrial process in which they are used.

Combined Effects of Endoxylanases and Reduced Water Levels in Pasta Production

ABSTRACTThe combined effects on pasta properties of 1) varying dosages of endoxylanases (EC 3.2.1.8) from Aspergillus aculeatus and Bacillus subtilis and 2) lower levels of water during pasta dough processing were studied. The A. aculeatus endoxylanase has high selectivity toward water‐extractable arabinoxylan (WE‐AX), whereas B. subtilis endoxylanase preferentially hydrolyzes water‐unextractable arabinoxylan (WU‐AX). Pasta was produced on a microscale (50.0 g) from the semolinas of both a strong (AC Navigator) and a moderately strong (AC Avonlea) durum wheat cultivar. The levels of added water in endoxylanase‐treated pastas were adjusted to obtain the same maximal farinograph consistencies as for the control pastas. The extruded pastas were dried with drying cycles at 40, 70, or 90°C. Apart from increasing levels of solubilized arabinoxylans, these treatments had little effect on the color, optimal cooking time, and firmness of the resulting pasta. High enzyme concentrations and low (40°C) drying temperature resulted in clearly or much less checked final products for the B. subtilis and A. aculeatus enzyme, respectively. Upon cooking, the enzymically formed low molecular weight arabinoxylans were retained better in the pasta strands than their equally low molecular weight arabinogalactan counterparts.

Synergy between enzymes involved in the degradation of insoluble wheat flour arabinoxylan

Two microbial endo-β-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 were examined for their ability to release ferulic acid (FA) from water-unextractable arabinoxylan (WU-AX) in the presence of a feruloyl esterase (FoFAE-II) from Fusarium oxysporum. WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 (XYLI) and a Sporotrichum thermophile family 11 (XYLA) endoxylanases. At 10 g/l arabinoxylan, enzyme concentrations ( K E values) needed to obtain half-maximal hydrolysis rates for FA release were 0.18 and 0.44 nM for the xylanases from T. aurantiacus and S. thermophile, respectively. Determination of V max/ K E revealed that the family 10 enzyme performed 4.3 times more efficiently than the family 11 enzyme in liberation of FA when a feruloyl esterase is present. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The results showed that the degradation of the xylan backbone was influenced strongest by the action of xylanases while the presence of the esterase mainly resulted in the release of ferulic acid from the produced short chain feruloylated xylo-oligosaccharides by the action of xylanases.

Variability in the Content of Water-Extractable and Water-Unextractable Non-Starch Polysaccharides in Rye Flour and Their Relationship to Baking Quality Parameters

The non-starch polysaccharide (NSP) content and composition of rye flours, differing in baking quality, were studied over three consecutive years at three locations. Rye flours were rich in water-extractable NSP and, consequently, rich in water-extractable arabinoxylans (WE-AX). A large variability of both components was observed in water-unextractable fraction, and especially, in the ratio of water-extractable to water-unextractable arabinoxylan content (WE/U-AX). The proportion of WE/U-AX in rye flour was found to be the primary factor contributing to both flour and bread quality parameters. However, the content of WE-AX correlated positively but not significantly, whereas water-unextractable (WU) counterpart was negatively correlated with quality attributes. The data showed that rye flour of high baking quality had a high ratio of WE/U-AX, which was also combined with high values of falling number and amylographic peak viscosity. Significant, positive correlations were also found between ratio of water extract viscosity to water-extractable arabinoxylan content (EV/WE-AX) and overall baking score and bread volume. Our results suggest that some AX populations, of specific structure and/or molecular weight, could be especially important for baking potential of rye flour.

Relative activity of two endoxylanases towards water-unextractable arabinoxylans in wheat bran

Destarched, deproteinised wheat bran contains ca 40% of water-unextractable arabinoxylan (WU-AX) which is solubilised more effectively by Bacillus subtilis endoxylanase (EC 3.2.1.8, XBS, ca. 41% of WU-AX) than by Aspergillus aculeatus endoxylanase (XAA, ca. 18%). While XBS preferentially converts WU-AX into enzymically solubilised arabinoxylan (ES-AX), XAA preferentially degrades water-extractable arabinoxylan and ES-AX. Arabinose to xylose ratios of ES-AX, released by XBS, decreased with increasing enzyme levels indicating an unexpected difference in their enzymic solubilisation which may be due to close packing of AX with low degree of substitution and/or limited solubility of the hydrolysed fragments. Wheat bran ES-AX released at low XBS concentrations had a higher relative peak molecular mass than those released at higher XBS concentrations. ES-AX invariably was of low relative molecular mass for all XAA concentrations tested. The results indicate that XBS is useful for the conversion of insoluble fibre to soluble fibre.

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan

Microbial endo-β-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l −1 arabinoxylan, enzyme concentrations ( K E values) needed to obtain half-maximal hydrolysis rates ( V max values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of V max/ K E revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX 2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX 3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.