Xylan is the major hemicellulose present within most dedicated energy crops and agricultural residues, comprising between 20 % and 30 % of cell wall material. Xylan is a polysaccharide composed of β-1,4-linked xylose residues with side groups that include arabinose, glucuronic acid (GlcA), and/or acetate. Additional structural complexity exists due to potential additional modifications on these side groups (additional sugar groups, acetylation, feruloylation, and methylation at the 4-hydroxy position of glucuronic acid (4-OMe-GlcA)) [1]. In general, these side groups block enzymatic conversion of polysaccharides or oligosaccharides to monosaccharides. Plant cell wall xylan is challenging to hydrolyze with enzymes because of this structural complexity [2], where each substituent linkage type requires its own enzyme [3], [4]. Incomplete hydrolysis limits monosaccharide yield, making a portion of the xylose unavailable for microbial conversion. The enzymatic hydrolysis of xylan to monosaccharides occurs by the combined action of β-xylanases (GH10 hydrolysis yields oligosaccharides with substitutions on the non-reducing end; GH11 hydrolysis yields oligosaccharides with internal substitutions), followed by the action of β-xylosidases that cleave linear xylooligosaccharides to xylose [4]. Further, partially digested xylan (i.e. xylan polymers and oligosaccharides) inhibits cellulases, which can increase enzyme loading and/or decrease glucose yield obtained from hydrolysis of cellulose [5], [6], [7], [8].

The glucuronosyl substitution on xylan resists acidic hydrolysis of the xylan polymer [9]. Enzymatic targeting of glucuronosyl substitutions relies on α-glucuronidases (EC 3.2.1.131; Agu); these glycoside hydrolases (GH) are classified in two families by the Carbohydrate-Active enZYme (CAZy) database (http://www.cazy.org/): GH67 (26 characterized in CAZy database) and GH115 (nine characterized in CAZy database). GH67 family α-glucuronidases only remove the glucuronosyl group attached to the terminal residue at the non-reducing end of xylooligosaccharides (XOS), whereas GH115 enzymes act on α-(1→2)-linked GlcA-containing polysaccharides and oligosaccharides. GH115 α-glucuronidases are able to hydrolyze glucuronic acids from internal sites in polysaccharide or oligosaccharide chains, as well as glucuronosyl groups on the terminal non-reducing end residue. The ability to cleave glucuronic acids from internal positions in a xylan chain should be beneficial, as a previous study shows that residual oligosaccharides from unhydrolyzed biomass samples are highly represented by glucuronic acid-containing oligosaccharides [10] and GH115 enzymes will not rely on β-xylanase and β-xylosidase activity to produce a non-reducing end containing GlcA-oligosaccharide. Our goal is to identify α-glucuronidase enzymes that are compatible with the pH and temperature of saccharification cocktails. Of the nine characterized GH115 enzymes, seven are from bacterial sources [11], [12], [13], [14], [15], [16] and two reports are from fungal sources [17], [18] (Table 1). Interestingly, one bacterial GH115 is reported as active only on terminal 4-OMe-GlcA residues from decorated arabinogalactan [15]. In two recent reports of GH115 enzymes, kinetic constants are not reported; however, these studies used fungal sources from: Rasamsonia emersonii, along with a GH67 α-glucuronidase from the same organism, for mode of action with some pH and temperature dependence data [19]; and Penicillium subruescens for mode of action at a pH and temperature near the values for saccharification cocktails [20] (Table 1).

Therefore, to better hydrolyze residual oligosaccharides from biomass hydrolysis, having more GH115 enzymes available will be beneficial for conversion. One candidate to fill this role is a putative GH115 from a soil fungus, Coniochaeta ligniaria. In this study, we report the constitutive expression of the putative GH1115 gene (OIW23892.1), of C. ligniaria, using the native sequence in the native organism. Glucuronidase activity was shown on full-length beech xylan consistent with a GH115 family assignment. The enzyme, ClAgu115, was purified by multi-step standard protein separations. After purification of the enzyme, we report characteristics of ClAgu115 including: pH and temperature optima; temperature stability; and kinetic constants for the hydrolysis of three substrates, representing different characteristics that would be present during biomass hydrolysis (i.e. full-length xylan and a mixture of glucuronic acid-containing oligosaccharides containing either non-reducing end glucuronic acids or internal glucuronic acids).