Development of an automatable method for the measurement of endo-1,4-β-xylanase activity in barley malt and initial investigation into the relationship between endo-1,4-β-xylanase activity and wort viscosity
Introduction
In 2014, Hordeum vulgare L. (malting barley) was ranked 4th in cereal grains in terms of global production at 144 million tonnes (Food and Agriculture Organization of the United Nations (FAOSTAT), 2014). The majority of this usage is in the brewing industry where malted barley is one of the key ingredients for the production of beer or whiskey. In barley, the cell walls of the aleurone tissue are constituted primarily of arabinoxylan (Bacic and Stone, 1981) while the starchy endosperm cell walls are composed of ∼20% arabinoxylan with ∼75% of the remainder being made up of mixed linkage (1,3; 1,4)-β-glucan (Fincher, 1975). Traditionally, the brewing fitration issues associated with β-glucan have been more widely studied in brewing science (Narziss, 1993). However, the importance of arabinoxylan has also been demonstrated previously with respect to production issues such as poor mash separation, beer filterability and haze formation (Barrett et al., 1975; Coote and Kirsop, 1976; Li et al., 2015; Lu and Li, 2006). As endo-1,4-β-xylanase (xylanase, EC 3.2.1.8) is known to readily degrade the arabinoxylan present in barley (Debyser et al., 1997; Viëtor et al., 1994), it clearly has the potential to significantly effect both wort composition and also the processing efficiency and quality of the finished beer product.
A number of isoforms of barley xylanase have been purified previously and characterised (Benjavongkulchai and Spencer, 1986; Dashek and Chrispeels, 1977; Kanauchi et al., 2013; Slade et al., 1989). Reported molecular weights range from ∼29,000–67,000. Isoelectric points have been observed between 4.6 and 6.5, with a pH optima appearing to vary between ∼5.0–6.7. However, all previous studies seem to be in agreement that the predominant forms exhibit a broad pH range with >75% activity maintained from approximately pH 4.5–7. It had been reported that the largest of these enzymes (Mr = 61500) was an inactive form that needed to be processed by endogenous proteolytic machinery to produce active forms with lower molecular weight (Mr = 34,000 and 41,000) (Caspers et al., 2001), but in fact the larger isoform was later shown to be active following recombinant expression by Van Campenhout et al. (2007), a conclusion that has been independently confirmed through conventional protein purification techniques from barley by Kanauchi et al. (2013). Xylanase inhibitors of both TAXI (Goesaert et al., 2001) and XIP (Goesaert et al., 2003) type have been reported in barley but these have been shown only to inhibit microbial xylanases, having no measurable effect on the endogenous xylanases. This aspect has been reviewed in depth by Gusakov (2010).
The measurement of xylanase for cereal quality analysis was traditionally performed using either viscometric (Preece and MacDougall, 1958) or reducing sugar procedures (Benjavongkulchai and Spencer, 1986) until the advent of commercially available azurine dyed and cross-linked (AZCL) wheat arabinoxylan (McCleary, 1992) in the form of Xylazyme tablets (insoluble substrate) which has since found widespread use, particularly for the analysis of crude cereal extracts (Debyser et al., 1997; Fierens et al., 2008, 2007). Cürten et al. (2018), have recently described an ‘on-line’ xylanase assay involving the use of HPAEC-PAD where the enzymatic incubations take place in a standard autosampler with multiple injections to monitor the reaction time course. The current authors have previously reported the development of a ‘soluble substrate’ enzyme coupled assay procedure based on a novel colourimetric hexasaccharide substrate, 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-45-O-D-glucosyl-xylopentaoside, for the measurement of xylanase (XylX6 assay) (Mangan et al., 2017). The development of a new assay format employing this soluble substrate, that is suitable for the automated analysis of xylanase in barley malt extracts, is described herein. The application of this procedure to the measurement of xylanase in a small sample set of barley malts and demonstration of the apparent correlation between these activities and the viscosity of their resulting worts is also described.
Section snippets
Materials
The XylX6 reagent was obtained from Megazyme (K-XylX6-1 V) and contains lyophilised powder to be dissolved as a 5 mL solution of 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-45-O-glucosyl-xylopentaoside (3.3 mM) and GH43 β-xylosidase from Selenomonas ruminantium (20 U/mL) which is sufficient to perform 50 malt xylanase assays under the procedure described here. The control malt flour (lot number 61002A) from the Malt Beta-Glucanase/Lichenase Assay Kit (K-MBG4) was obtained from Megazyme – hereafter
Results and discussion
In order to develop a procedure for the measurement of xylanase in malt, two separate components required optimisation – the biochemical assay conditions and the malt extraction procedure. To obtain an extract containing sufficient xylanase activity to develop the assay parameters, conditions that were recently employed for the extraction of barley β-glucanase (Mangan et al., 2016) were used without optimisation. The effect of the XylX6 reagent concentration on the assay response was
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