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.
Comparison of endolytic hydrolases that depolymerize 1,4-β-D-mannan, 1,5-α-L-arabinan and 1,4-β-D-galactan.
McCleary, B. V. (1991). “Enzymes in Biomass Conversion”, (M. E. Himmel and G. F. Leatham, Eds.), ACS Symposium Series 460, Chapter 34, pp. 437-449. American Chemical Society, Washington.
Hydrolysis of mannan-type polysaccharides by β-mannanase is dependent on substitution on and within the main-chain as well as the source of the β-mannanase employed. Characterisation of reaction products can be used to define the sub-site binding requirements of the enzymes as well as the fine-structures of the polysaccharides. Action of endo-arabinanase and endo-galactanase on arabinans and arabinogalactans is described. Specific assays for endo-arabinanase and arabinan (in fruit-juice concentrates) are reported.
Measurement of endo-1,4-β-D-xylanase.
McCleary, B. V. (1992). “Xylans and Xylanases”, (J. Visser, G. Beldman, M. A. Kusters-van Someron and A. G. J. Voragen, Eds.), Progress in Biotechnology, Vol. 7, Elsevier, Science Publishers B. V., pp. 161-169.
Various procedures for the measurement of xylanase in fermentation broths, commercial enzyme mixtures, bread improver mixtures and feed samples are described. Problems associated with the routine use of reducing-sugar based methods axe highlighted and the advantages and limitations of viscometric and dye-labelled substrate procedures for measurement of trace levels of activity in feed samples are discussed.
New developments in the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase.
McCleary, B. V. & Monaghan, D. (2000). “Proceedings of the Second European Symposium on Enzymes in Grain Processing”, (M. Tenkanen, Ed.), VTT Information Service, pp. 31-38.
Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.
Analysis of feed enzymes.
McCleary, B. V. (2001). “Enzymes in Farm Animal Nutrition”, (M. Bedford and G. Partridge, Eds.), CAB International, pp. 85-107.
Enzymes are added to animal feed to increase its digestibility, to remove anti-nutritional factors, to improve the availability of components, and for environment reasons (Campbell and Bedford, 1992; Walsh et al., 1993). A wide-variety of carbohydrase, protease, phytase and lipase enzymes find use in animal feeds. In monogastric diets, enzyme activity must be sufficiently high to allow for the relatively short transit time. Also, the enzyme employed must be able to resist unfavourable conditions that may be experienced in feed preparation (e.g. extrusion and pelleting) and that exist in the gastrointestinal tract. Measurement of trace levels of enzymes in animal feed mixtures is difficult. Independent of the enzyme studied, many of the problems experienced are similar; namely, low levels of activity, extraction problems inactivation during feed preparation, non-specific binding to other feed components and inhibition by specific feed-derived inhibitors, e.g. specific xylanase inhibitors in wheat flour (Debyser et al., 1999).
Measurement of polysaccharide degrading enzymes using chromogenic and colorimetric substrates.
McCleary, B. V. (1991). Chemistry in Australia, September, 398-401.
Enzymic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material. In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall degrading enzymes are used to destroy the three-dimensional structure and water binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial process.
Optimising the response.
Acamovic, T. & McCleary, B. V. (1996). Feed Mix, 4, 14-19.
A fine balance exists between enzyme activity and the adverse effects associated with feed processing. Accurate estimation of enzyme activity in the feed is a pre-requisite to optimising the response.
Xylanase B from the hyperthermophile Thermotoga maritima as an indicator for temperature gradients in high pressure high temperature processing.
Vervoort, L., Van der Plancken, I., Grauwet, T., Verjans, P., Courtin, C. M., Hendrickx, M. E. & Van Loey, A. (2011). Innovative Food Science & Emerging Technologies, 12(2), 187-196.
Within the scope of high pressure food sterilization, an important issue that should be taken into account in refining process and equipment design is the time- and position-dependent temperature gradient that exists throughout the pressure vessel and the product load. Since enzymes from thermophilic microorganisms show good prospects for the development of indicators to map out the temperature non-uniformity in high pressure high temperature (HPHT) processing, in this work, the potential of xylanase B from Thermotoga maritima (XTMB) was investigated. Its inactivation at isothermal–isobaric conditions was best described by a first-order model. The pressure dependence of the D values was negligible at HPHT, the temperature dependence however was substantial. The Thermal Death Time (TDT) model, and its corresponding parameters, describing this large temperature dependence were successfully validated under dynamic processing conditions, relevant for industrial HPHT applications.
Despite extensive research progress on high pressure high temperature (HPHT) processing as a new food sterilization technique, food industry should be aware of a possible non-uniform temperature distribution, occurring in the pressure vessel and its consequence for the quality and safety of treated products. Since direct measurement of the temperature distribution is not feasible with the measuring devices currently available and constructive computation of the temperature profile by numerical simulation is inadequate, the development of specific temperature-sensitive wireless sensors, or pressure–temperature–time indicators (pTTIs) can be put forward. In this work, xylanase B from Thermotoga maritima (XTMB) was evaluated as a potential enzymatic indicator for mapping the temperature non-uniformity in HPHT processing.
Identification of multiple highly similar XIP-type xylanase inhibitor genes in hexaploid wheat.
Takahashi-Ando, N., Inaba, M., Ohsato, S., Igawa, T., Usami, R. & Kimura, M. (2007). Biochemical and Biophysical Research Communications, 360(4), 880-884.
In hexaploid wheat, Xip-I is the only XIP-type xylanase inhibitor gene whose expression and function have been characterized in detail. Here we demonstrate the existence of new XIP-type genes with the identification of Xip-R1 and Xip-R2 in the root cDNAs. Southern blot analysis with the Xip-R1probe revealed that XIP-type genes comprised a significantly greater gene family than previously speculated on in studies with the Xip-I probe. The transcript level of Xip-R genes was increased upon an inoculation with Erysiphe graminis in the leaves, but not with Fusarium graminearum in the spikelets. RT-PCR with the RNA samples followed by extensive sequencing of the cloned amplified products revealed the presence of 12 highly similar Xip-R genes. Among these genes, Xip-R1 was the only predominant Xip-R family member induced to express in response to E. graminis. XIP-R1 was located in the apoplastic space and inhibited family 11 xylanases, but the protein did not show chitinolytic activity. These results suggest that hexaploid wheat has a large family of XIPs in its genome, but that only some of them are expressed for plant defense in limited tissues.