Background

Xylan is one of the most abundant polysaccharides in nature, having a highly complex tertiary structure organized as a linear backbone of D-xylose with β-1,4-linkages, usually branched with other sugars such as α-arabinose and/or organic acids [1,2]. Due to its heterogeneous architecture, xylan degradation is very challenging, but the process can be effectively carried out by xylanases that hydrolyze β-1,4-glycosidic bonds. Xylanases are glycoside hydrolases (GHs) expressed by various organisms, including bacteria, yeast, fungi, and crustaceans [3]. The genomic, structural, and functional information of xylanase can be found in the carbohydrate-active enzyme database (CAZy) [4]. Xylanases are generally associated with GH families 5, 7, 8, 9, 10, 11, 12, 16, 26, 30, 43, 44, 51, and 62 [4], hydrolyzing xylan substrates according to two modes of action: retention and inversion [5,6]. This wide spectrum of physicochemical properties, substrate specificities, and catalytic mechanisms makes xylanases highly versatile for numerous applications across industrial and biotechnological sectors [7]. Nowadays, their use spans pulp bleaching in pulp and paper industries [8], bakery and food processing industries [9,10], prebiotics and potential anticancer agents in pharmaceutical sectors [11,12], and lignocellulosic biorefining [13].

Numerous methods have been developed to measure endo-xylanase activity. These assays differ not only in assay conditions (temperature, incubation time, substrate, etc.) but also in the quantification method of enzymatic activity. For decades, the preferred method was the measurement of reducing sugars liberated from the polymer through the 3,5-dinitrosalicylic acid (DNS) method [14] or the Nelson/Somogyi method [15,16]. However, both procedures present some limitations, especially in terms of a lack of stoichiometric relation of the color response and turbidity [17]; in addition, these assays are nonspecific, and other enzymatic activities can be involved in the product determination, such as β-xylosidase and α-glucuronidase [18]. Viscosimetric methods provide benefits in terms of specificity and sensitivity, but they are also time-consuming and show a limited throughput of just a few reactions per day [18]. Nevertheless, fermentation broths or complex mixtures are generally composed of a cocktail of endo-xylanase and other glycosidases, including α-L-arabinofuranosidase and β-xylosidase, making the process of quantification quite challenging. To overcome these limitations, soluble dyed polysaccharides, such as Azo-Xylan or Azo-wheat arabinoxylan, are extensively used and give the possibility to measure enzymatic activity even in preparations with high levels of reducing sugars [18]. The Azo-Xylan assay is specific for endo-1,4-β-D-xylanase activity, and the protocol can be carried out following the manufacturer’s instructions (Megazyme, Bray, Ireland). In principle, to obtain Azo-Xylan, birchwood xylan is first purified to remove starch and then it is chemically dyed with Remazol Brilliant Blue R™ to an extent of approximately one dye molecule per 30 sugar residues. After incubation of Azo-Xylan with endo-xylanases, the substrate is depolymerized to produce low-molecular-weight water-soluble fragments (Figure 1), which remain in solution, resulting in a colored supernatant that can be directly correlated to endo-xylanase activity by reference to a standard curve (Megazyme, Bray, Ireland). Herein, we adapted the classical version of this protocol to measure the endo-xylanase activity of thermostable xylanases secreted by different microbial consortia, aiming to the determination of enzyme temperature and pH optima, thermostability, and shelf life [19,20]. The modifications can help in the determination of the biochemical parameters of enzymes in complex mixtures and can enhance the possibility of obtaining a complete characterization in a rapid and cheap way.

Figure 1. Representation of the Azo-Xylan molecule. Adapted from the picture available at Megazyme website (https://prod-media.megazyme.com/media/pdf/71/fd/ce/enzyme-substrates-brochure.pdf).