GH30-7 Endoxylanase C from the Filamentous Fungus .

Glycoside hydrolase family 30 subfamily 7 (GH30-7) enzymes include various types of xylanases, such as glucuronoxylanase, endoxylanase, xylobiohydrolase, and reducing-end xylose-releasing exoxylanase. Here, we characterized the mode of action and gene expression of the GH30-7 endoxylanase from the cellulolytic fungus (Xyn30C). Xyn30C has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal cellulose binding module 1, whose cellulose-binding ability has been confirmed. Sequence alignment of GH30-7 xylanases exhibited that Xyn30C has a conserved Phe residue at the position corresponding to a conserved Arg residue in GH30-7 glucuronoxylanases, which is required for the recognition of the 4--methyl-α-d-glucuronic acid (MeGlcA) substituent. Xyn30C degraded both glucuronoxylan and arabinoxylan with similar kinetic constants and mainly produced linear xylooligosaccharides (XOSs) with 2 to 3 degrees of polymerization, in an endo manner. Notably, the hydrolysis of glucuronoxylan caused an accumulation of 2-(MeGlcA)-xylobiose (UX). The production of this acidic XOS is likely to proceed via multistep reactions by putative glucuronoxylanase activity that produces 2-(MeGlcA)-XOSs (X UX,  ≥ 0) in the initial stages of the hydrolysis and by specific release of UX from a mixture containing X UX. Our results suggest that the unique endoxylanase activity of Xyn30C may be applicable to the production of linear and acidic XOSs. The gene was located adjacent to the putative GH62 arabinofuranosidase gene () in the genome. The expression of both genes was induced by cellulose. The results suggest that Xyn30C may be involved in xylan removal in the hydrolysis of lignocellulose by the cellulolytic system. Xylooligosaccharides (XOSs), which are composed of xylose units with a β-1,4 linkage, have recently gained interest as prebiotics in the food and feed industry. Apart from linear XOSs, branched XOSs decorated with a substituent such as methyl glucuronic acid and arabinose also have potential applications. Endoxylanase is a promising tool in producing XOSs from xylan. The structural variety of XOSs generated depends on the substrate specificity of the enzyme as well as the distribution of the substituents in xylan. Thus, the exploration of endoxylanases with novel specificities is expected to be useful in the provision of a series of XOSs. In this study, the endoxylanase Xyn30C from was characterized as a unique glycoside hydrolase belonging to the family GH30-7, which specifically releases 2-(4--methyl-α-d-glucuronosyl)-xylobiose from hardwood xylan. This study provides new insights into the production of linear and branched XOSs by GH30-7 endoxylanase.