Xylobiose is a good prebiotic and is used as an additive in the food and feed industries. The fungal strain Aspergillus fumigatus FC2-2, isolated from soil, secretes xylanases that can convert birchwood xylan and extracted xylan from sugarcane bagasse to xylobiose as the main component. A xylanase AfxynA produced by the strain FC2-2 was purified and characterized. The AfxynA hydrolyzed the xylooligosaccharides, birchwood xylan, and sugarcane bagasse xylan, mainly into xylobiose. The half-life of the purified AfxynA at 50 degrees C and 55 degrees C was 4207.9 min and 1001.6 min, respectively. The N-terminal sequence of the purified AfxynA was determined to be AGTPSSTG, and the AfxynA was found to belong to the glycosyl hydrolase family 11. The cDNA gene encoding AfxynA was cloned and overexpressed in Escherichia coli, and the gene was site-directed mutated to replace the codons encoding Asn70, Arg121, or Gln135 by Ala. The mutant enzymes N70A, R121A, and Q135A showed reduced enzyme activity in comparison with the native enzyme. More interestingly, the hydrolysis product of birchwood xylan by these mutant xylanases showed less proportion of xylobiose than that by the native enzyme. The above results indicated that N70, R121, and Q135 of AfxynA are required for xylanase activity and formation of xylobiose as a major product. (C) 2015 Elsevier Ltd. All rights reserved.