Novel substrates for the automated and manual assay of endo-1,4-β-xylanase.
Mangan, D., Cornaggia, C., Liadova, A., McCormack, N., Ivory, R., McKie, V. A., Ormerod, A. & McCleary, D. V. (2017). Carbohydrate Research, 445, 14-22.
endo-1,4-β-Xylanase (EC 126.96.36.199) 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.
Preparation of arabinoxylobiose from rye xylan using family 10 Aspergillus aculeatus endo-1,4-β-D-xylanase.
Rantanen, H., Virkki, L., Tuomainen, P., Kabel, M., Schols, H. & Tenkanen, M. (2007). Carbohydrate Polymers, 68(2), 350-359.
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.
In vitro fermentation kinetics and end-products of cereal arabinoxylans and (1,3;1,4)-β-glucans by porcine faeces.
Williams, B. A., Mikkelsen, D., Le Paih, L. & Gidley, M. J. (2011). Journal of cereal science, 53(1), 53-58.
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.
Effects of wheat-bran arabinoxylan as partial flour replacer on bread properties.
Koegelenberg, D. & Chimphango, A. F. (2017). Food Chemistry, 221, 1606-1613.
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.
Laccase/TEMPO oxidation in the production of mechanically strong arabinoxylan and glucomannan aerogels.
Parikka, K., Nikkilä, I., Pitkänen, L., Ghafar, A., Sontag-Strohm, T. & Tenkanen, M. (2017). Carbohydrate Polymers, 175, 377-386.
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.