Arabinoxylan (Wheat Flour; High Viscosity)

endo-1,4-β-Xylanase (EC 3.2.1.8) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-4⁵-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.

In this study, an endo-1,4-β-xylanase was purified from wheat malt following the procedures of ammonium sulfate precipitation, cation-exchange chromatography, and two-step anion-exchange chromatography. The purified endo-1,4-β-xylanase had a specific activity of 3.94 u/mg, demonstrating a weight average molecular weight (Mw) of approximately 58,000 Da. After LC-MS/MS (Liquid chromatography-tandem mass spectrometry) identification, the purified enzyme had the highest matching degree with a GH10 (Glycoside Hydrolase 10) domain-containing protein from wheat, there were 23 match peptides with a score above the threshold and the prot-cover was 45.5%. The resulting purified enzyme was used to investigate its degradation ability on high viscosity wheat-derived water-extractable arabinoxylan (WEAX). Degradation experiments confirmed that the purified enzyme was a true endo-acting enzyme, which could degrade large WEAX into smaller WEAX. The average degree of polymerization (avDP) and the viscosity of WEAX decreased with the increasing reaction time. The enzyme could degrade a small amount of WEAX into arabinoxylan-oligosaccharides (AXOS) with a degree of polymerization of 2-6, but no monosaccharide was produced. The degradation occurred rapidly in the first 3.5 h and decreased with the further prolongation of reaction time.

We examined the initial attachment of E. coli to abiotic surfaces conditioned with cereal extracts. The extracts were water-soluble fractions prepared from flours of barley, quinoa, rice, and wheat. Strains used were E. coli ATCC 8739, E. coli NBRC 3301, E. coli NBRC 3302, E. coli NBRC 13168, E. coli NBRC 13891, and E. coli O157:H7 sakai. When surfaces of glass and stainless steel were conditioned at 25°C for 30 min with 0.5% cereal extracts, significantly lower numbers of E. coli cells attached to the conditioned surfaces than unconditioned ones, irrespective of strains used. The highest activity in reduction of the number of E. coli cells attached to the abiotic surfaces was found in the wheat extract. The suppressive activity was stable after treatments of the extract by autoclave and enzymatic digestion with α-amylase and proteinase K. We purified the active compound by ammonium sulfate fractionation and gel filtration with HiPrep 16/60 Sephacryl S-200 HR after the enzymatic treatments. The purified compound showed an average molecular mass of about 300 kDa by light-scattering measurements. Analyses of its components indicated that the active compound was arabinoxylan; the molar ratios were 1.0 (arabinose) to 2.46 (xylose). Commercially available arabinoxylan (average molecular mass: 370 kDa) also showed the similar activity. To our knowledge, this is the first report on a dietary fiber from cereals which suppresses the initial attachment of E. coli to abiotic surfaces.

Backgound: Xylan is the second most abundant plant cell wall polysaccharide after cellulose with α-L-arabinofuranose (L-Araf) as one of the major side substituents. Capacity to degrade xylan is characteristic of many plant pathogens; and corresponding enzymes that debranch arabinoxylan provide tools to tailor xylan functionality or permit its full hydrolysis. Method: Three GH62_2 family α-arabinofuranosidases (Abfs) from plant pathogenic fungi, NhaAbf62A from Nectria haematococca, SreAbf62A from Sporisorium reilianum and GzeAbf62A from Gibberella zeae, were recombinantly produced in Escherichia coli. Their biochemical properties and substrate specificities were characterized in detail. Particularly with ¹H NMR, the regioselectivity and debranching preference of the three Abfs were directly compared. Results: The activities of selected Abfs towards arabinoxylan were all optimal at pH 6.5. Their preferred substrates were wheat arabinoxylan, followed by soluble oat spelt xylan. The Abfs displayed selectivity towards either α-(1 → 2) or α-(1 → 3)-L-Araf mono-substituents in arabinoxylan. Specifically, SreAbf62A and GzeAbf62A removed m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substituents with a similar rates, whereas NhaAbf62A released m-α-(1→ 3)-L-Araf 1.9 times faster than m-α-(1 → 2)-L-Araf. Major conclusions: Building upon the known selectivity of GH62 family α-arabinofuranosidases towards L-Araf mono-substituents in xylans, the current study uncovers enzyme-dependent preferences towards m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substitutions. Comparative sequence-structure analyses of Abfs identified an arginine residue in the xylose binding +2R subsite that was correlated to the observed enzyme-dependent L-Araf debranching preferences. General significance: This study expands the limited pool of characterized GH62 Abfs particularly those from plant pathogenic fungi, and provides biochemical details and methodology to evaluate regioselectivity within this glycoside hydrolase family.

Background: Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals. Results: A novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65°C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25°C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60°C for 60 h, and its half-lives at 65°C and 70°C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-D-cellobioside (pNPC), and p-nitrophenyl-β-D-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%. Conclusions: XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.

Effects on physical properties of white bread of adding crude (E1) and partially purified (E2) arabinoxylans (AX) from wheat bran to partially replace flour during baking, were investigated to identify optimal dosage. The E1 and E2 had molecular weights of 620,000 and 470,000 Da with arabinose to xylose ratio of 0.7 and 0.6, respectively. However, ferulic acid of 1.5 mg/100 g, was detectable only in E1. The AXs were added to 100 g white bread formulae at dosages of 0.8–1.2% with flour removal of 2–3% (w/w). The dough increased water absorption by 2% in the specified dosage range. An optimum dosage of 0.8% with 2.5% flour removal maintained similar weight, volume, height and firmness as standard white bread. At this dosage, AX addition in white bread holds both increased health and economic benefits because of combined roles as soluble dietary fibre and flour replacer.

New wheat arabinoxylan and konjac glucomannan hydrogels and aerogels were prepared by hemiacetal crosslinking induced by laccase/TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) -catalysed oxidation, which selectively converts the primary hydroxyl groups to aldehydes. The degree of oxidation of the product aldehydes was ca. 10% of the total carbohydrates of the polysaccharides, and the determination of storage and viscous moduli of the oxidised samples showed that they had formed true hydrogels. Two freezing methods for the hydrogels, conventional freezing and ice crystal templating, were investigated for aerogel production, the ice crystal templated products especially were mechanically strong in compression test against the ice crystals’ growth direction. The compressive moduli were ca. 1200 kPa for wheat arabinoxylan aerogels and ca. 650 kPa for konjac glucomannan aerogels. A morphological study with a scanning electron microscope revealed the inner structure of the aerogels. Ice crystal templated konjac glucomannan aerogel formed round pores with a diameter of ca. 50-100 µm. The arabinoxylan aerogel consisted of long and narrow pores with a length of a few hundred µm and width of 50-100 µm, which had formed in the direction of the ice crystals’ formation. Konjac glucomannan and wheat arabinoxylan are approved food-grade materials, and wheat arabinoxylan is particularly interesting because it can be obtained from cereal processing side streams − thus, these novel products have potential in various applications, including the food, food packaging, and pharmacological fields.

Purified and semi-purified polysaccharides characteristic of cereals were fermented in vitro with a pig faecal inoculum, using the cumulative gas production technique, to examine the kinetics and end-products of fermentation after 48 h. It was shown that arabinoxylan and mixed linkage (1,3;1,4) β-glucan were rapidly fermented if soluble, while less soluble substrates (insoluble arabinoxylan, maize and wheat starch granules, and bacterial cellulose) were more slowly fermented. Relevant monosaccharides were fermented at very similar rates to soluble polymeric arabinoxylan and β-glucan, showing that depolymerisation was not a limiting step, in contrast to some previous studies. Bacterial cellulose is shown to be a useful model substrate for fermentation of plant cellulose which is difficult to obtain without harsh chemical treatments. Fermentation end-products were related to kinetics, with slow carbohydrate fermentation resulting in increased protein fermentation. Ratios of short-chain fatty acid products were similar for all arabinoxylan and β-glucan substrates.

Commercial xylanase preparation Shearzyme^®, which contains the glycoside hydrolase family 10 endo-1,4-β-D-xylanase from Aspergillus aculeatus, was used to prepare short-chain arabinoxylo-oligosaccharides (AXOS) from rye arabinoxylan (AX). A major AXOS was formed as a hydrolysis product. Longer AXOS were also produced as minor products. The pure GH10 xylanase from A. aculeatus was used as a comparison to ensure that the formed AXOS were consequence of the endoxylanase‘s function instead of some side enzymes present in Shearzyme. The major AXOS was purified and the structure confirmed with various analysis methods (TLC, HPAEC-PAD, MALDI-TOF-MS, and one- and two-dimensional NMR spectroscopy with nano-probe) as α-L-Araf-(1→3)-β-D-Xylp-(1→4)-D-Xylp (arabinoxylobiose). This is the first report on ¹³C NMR data of pure arabinoxylobiose. The yield of arabinoxylobiose was 12% from the quantified hydrolysis products. In conclusion, GH10 endoxylanase from A. aculeatus is thus able to cut efficiently the xylosidic linkage next to the arabinofuranosyl-substituted xylose unit which is not typical for all the GH10 endoxylanases. Interestingly, pure A. aculeatus xylanase showed notably activity towards p-nitrophenyl-β-D xylopyranose. In previously studies longer AXOS have been produced with Shearzyme but the formation of short-chain AXOS by A. aculeatus GH10 xylanase has not been studied before.