Synergy of GH67 and GH115 α-1,2-glucuronidases with Penicillium subrubescens endoxylanases to stimulate xylooligosaccharide production

A primary substitution of the plant cell wall hemicellulosic polysaccharide xylan is (4-O-methyl-)d-glucuronic acid, which hinders the endoxylanases (XLNs) degradation of xylan for the production of valuable xylooligosaccharides (XOS). In this context, α-1,2-glucuronidase (AGU) plays a critical role in hydrolyzing the α-(1→2)-glycosidic linkages between 4-O-methyl-d-glucuronic acid and xylosyl residues in xylan, thereby enhancing XOS production by XLNs. However, AGUs have been relatively poorly studied, and insufficient and incomplete data on their biochemical properties, substrate specificity, and product profiling has limited their application. Here, we cloned, heterologously produced, purified and functionally characterized an AGU from Aspergillus niger (AnAguA) and another AGU from Penicillium subrubescens (PsAguB), belonging to Glycoside Hydrolase family 67 (GH67) and 115 (GH115), respectively, in the Carbohydrate-Active enZyme database. Results showed that neither AGU released 4-O-methyl-d-glucuronic acid from polymeric beech wood glucuronoxylan (BeWX). However, we found that from BeWX pre-digested with GH10 or GH11 XLNs from P. subrubescens (PsXlnA and PsXlnF, respectively), AnAguA released 4-O-methyl-d-glucuronic acid only from the non-reducing end of glucuronoxylan oligosaccharide, whereas PsAguB released 4-O-methyl-d-glucuronic acid from glucuronoxylan oligosaccharides regardless of the xylosyl substitution position. Furthermore, we demonstrated that enhancement of XOS release by adding AGUs to various combinations of GH10 (PsXlnA-C) and GH11 (PsXlnD-F, PsXlnH-I) XLNs from P. subrubescens varied based on the AGU-XLN combination. The combination of AnAguA with PsXlnA was the most effective, achieving at least a 3-fold increase in the release of XOS with a degree of polymerization of 5-7 compared to using PsXlnA alone.