A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase

Barry V Mccleary,Paraic Mcgeough

doi:10.1007/s12010-015-1803-z

Abstract

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.

Highlights

Xylan is one of the most abundant polysaccharides in nature
Since the nature of the xylan substrate used in the procedure for the assay of endo-xylanase may affect the colour response obtained from reducing end groups produced during the enzyme reaction, it is important to prepare the xylose standard in the presence of the substrate
The effect of beechwood glucuronoxylan, birchwood glucuronoxylan and wheat flour arabinoxylan on the colour response from xylose and xylobiose using the dinitrosalicylic acid (DNS) reagent is shown in Fig. 2a, b

Summary

Introduction

Xylan is one of the most abundant polysaccharides in nature. It consists of a main chain of β1,4-linked D-xylopyranosyl residue which is variously substituted by residues of 4-O-methylD-glucuronic acid, D-glucuronic acid or L-arabinofuranose and in some cases is esterifiedAppl Biochem Biotechnol (2015) 177:1152–1163 by acetyl groups [1]. Interest in xylan degradation spans a broad range of industries including the paper pulp industry where endo-xylanase has been successfully employed in cellulose pulp bleaching [5], improving the digestion of cereal-based feeds for chickens and pigs [6], improving the nutritional quality and digestibility of ruminant fodder [7] and improving the bread-making quality of wheat flours [8]. They are finding use in the production of xylosaccharides (XOS) and arabino-xylo-oligosaccharides (AXOS) for use as prebiotics in human nutrition [9]

Methods

Results

Conclusion