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 188.8.131.52) 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.
A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase.
McCleary, B. V. & McGeough, P. (2015). Appl. Biochem. Biotechnol., 177(5), 1152-1163.
The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.
Purification and Characterization of a Thermostable β-mannanase from Bacillus subtilis BE-91: Potential Application in Inflammatory Diseases.
Cheng, L., Duan, S., Feng, X., Zheng, K., Yang, Q. & Liu, Z. (2016). BioMed Research International, Article ID 6380147.
β-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the β-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a “two-step” method comprising ultrafiltration and gel chromatography. The purified β-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5-7.0. The β-mannanase activity was significantly enhanced in the presence of Mn+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+ and strongly inhibited by Ba2+, and Pb2+. Km and Vmax values for locust bean gum were 7.14 mg/mL and 107.5 μmol/min/mL versus 1.749 mg/mL and 33.45 µmol/min/mL for Konjac glucomannan, respectively. Therefore, β-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the β-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes.
Immobilization and stabilization of commercial β-1,4-endoxylanase DepolTM 333MDP by multipoint covalent attachment for xylan hydrolysis: Production of prebiotics (xylo-oligosaccharides).
Martins de Oliveira, S., Moreno-Perez, S., Romero-Fernández, M., Fernandez-Lorente, G., Rocha-Martin, J. & Guisan, J. M. (2017). Biocatalysis and Biotransformation, 1-10.
The commercial enzyme DepolTM 333MDP (D333MDP) was immobilized by multipoint covalent attachment onto 10% cross-linked agarose beads support highly activated with aldehyde groups. The enzyme immobilization process was very efficient, retaining 86% of its initial catalytic activity. Thermal stability of the immobilized D333MDP biocatalysts varied according to the incubation time of the enzyme-support. The optimal immobilized biocatalyst was produced after 24 h of incubation under alkaline conditions and longer incubation times resulted in a loss of stability. The optimal immobilized biocatalyst was 60- and 50-fold more stable at pH 5.5 and pH 7 at 50°C than the soluble enzyme, respectively. Activity and stability at pH 5.5 were enhanced when the optimal immobilized biocatalyst was modified by chemical amination of the enzyme surface. The chemical amination of the immobilized enzyme surface was 5-fold more stable at pH 5.5 and 50°C compared with the unmodified immobilized biocatalyst. The best immobilized biocatalysts (containing 100 UI/g of support) were evaluated in the beechwood xylan hydrolysis reaction at 50°C and pH 5.5. 80% of the reducing sugars were released after 6 h of hydrolysis with the aminated biocatalyst. Xylan hydrolysis reaction with the aminated biocatalyst was 80% faster than with the non-aminated one. The final composition of the xylooligosaccharides (XOS) obtained was identified and quantified by HPAEC-PAD which showed it was composed of 90% of xylobiose and 5% of xylotriose and xylose. The aminated immobilized-stabilized biocatalyst was used for four cycles of hydrolysis with no loss of catalytic activity, resulting in highly active and stable derivative suitable for industrial processes.
Improvement of the catalytic characteristics of a salt-tolerant GH10 xylanase from Streptomyce rochei L10904.
Li, Q., Sun, B., Li, X., Xiong, K., Xu, Y., Yang, R., Hou, J. & Teng, C. (2017). International Journal of Biological Macromolecules, In Press.
A GH10 xylanase Srxyn10 from Streptomyce rochei L10904, and its truncated derivative, Srxyn10M, were investigated. Both displayed great salt-tolerant ability, retaining more than 95% and 91% activity after incubation at 37°C for 1 h in 3.0 M and 5.0 M NaCl, respectively. They exhibited a special hydrolytic property of forming xylobiose as the major product and produced fewer xylose compounds when combined with a reported xylanase while digesting corncob xylans. The mutant, Srxyn10M, was constructed from Srxyn10 by deleting the C-terminal carbohydrate-binding module. It possessed a 3.26-fold higher specific activity on beechwood xylan than Srxyn10. Moreover, Srxyn10M showed greater substrate affinity and catalytic efficiency than Srxyn10 when beechwood xylan, birchwood xylan, and oat-spelt xylan were used as substrates. The thermostability was also greatly improved. Therefore, the application potential was markedly enhanced by the improvement of these properties.
Influence of viscosity on the growth of human gut microbiota.
Tamargo, A., Cueva, C., Álvarez, M. D., Herranz, B., Bartolomé, B., Moreno-Arribas, M. V. & Laguna, L. (2017). Food Hydrocolloids, In Press.
Numerous studies support the beneficial effects of dietary fibre. It is well known that fibre increases viscosity at intestinal level. Therefore, the effects of fibre on gut microbiota could be due not only by its intestinal bacteria fermentation but also to the increase in viscosity by itself. The aim of this study was to evaluate the effect of viscosity on the growth of gut microbiota at physiological conditions. For this purpose, four compartments from a gastrointestinal simulator (simgi®) were filled with Gut Nutrient Medium (GNM) plus different agar concentrations (0, 0.30, 0.45 and 0.60%), inoculated with faecal microbiota, and incubated 48 h under anaerobic conditions. Samples were collected at three time points (0, 24 h and 48 h) for representative intestinal bacterial enumeration and rheological characterization. Incubation of GNM gels with faecal microbiota changed the medium viscosity over time, even with constant conditions (temperature and pH). In such way that, in absence of agar (low viscosity), viscosity slightly increased over time; however, in viscous mediums, viscosity decreased over time. In relation to the growth of gut microbiota, results showed that viscosity favoured the growth of total anaerobes and Clostridium spp.; in contrast, total number of aerobes and members of the genus Enterococcus correlated negatively with viscosity increment. In conclusion, changes in intestinal viscosity seem to selectively modify microbiota composition. This is a pioneer work to understand the effect of food viscosity in the gastrointestinal system, showing that viscosity is an important factor itself to condition the growth of different bacteria’s groups.