Xylan, the most abundant hemicellulose, is a linear polymer of 1,4-beta-linked xylose residues, which can be substituted by methylated glucuronic acid, arabinofuranose or acetyl groups.  Endo-1,4-beta-xylanases (EC 3.2.1.8) are glycoside hydrolases capable of digesting the internal 1,4-beta-D-xylosidic linkages of xylan and xylosaccharides.  Other hemicellulases, such as α-glucuronidases, arabinofuranosidases and acetyl xylan esterases, cleave the various side chains from the xylose backbone. Four fungal endo-1,4-beta-xylanases were expressed in Aspergillus niger using the Gateway system. Two genes encode family 10 glycoside hydrolases, XynGH10-1 and XynGH10-2, and the other two genes code for family 11 glycoside hydrolases, XynGH11-1 and XynGH11-2.  XynGH10-2 possesses a type I carbohydrate-binding domain.  ClustalX alignments and protein modeling identified the active site residues and predicted their tertiary structures.  Optimal activity was obtained at pH 3.4 and 50°C with XynGH10-1, pH 4.5 and 70°C with XynGH10-2, pH 4.5 and 37°C with XynGH11-1 and pH 6 and 55°C with XynGH11-2.  Studies of the products released during the degradation of four types of xylan revealed significant differences in catalytic properties between the family 10 and the family 11 xylanases.  Major products of digestion by each xylanase on 4 types of xylan as well as unsubstituted xylo-oligomers were identified using thin layer chromatography and mass spectrometry.  Combination of these xylanases with arabinofuranosidase and α-glucuronidase showed synergy on xylan, with this effect varying according to the source of xylan and therefore its side chain composition. We plan to continue to examine synergism with these and other classes of hemicellulases.