A gluco-oligosaccharide oxidase (GOOX) from Acremonium strictum type strain CBS 346.70 was cloned and expressed in Pichia pastoris. The recombinant protein, GOOX-VN, contained fifteen amino acid substitutions compared with the previously reported A. strictum GOOX. These two enzymes share 97% sequence identity; however, only GOOX-VN oxidized xylose, galactose, and N-acetylglucosamine. Besides monosaccharides, GOOX-VN oxidized xylo-oligosaccharides, including xylobiose and xylotriose with similar catalytic efficiency as for cello-oligosaccharides. Of three mutant enzymes that were created in GOOX-VN to improve substrate specificity, Y300A and Y300N doubled k(cat) values for monosaccharide and oligosaccharide substrates. With this novel substrate specificity, GOOX-VN and its variants are particularly valuable for oxidative modification of cello-and xylo-oligosaccharides. Biotechnol. Bioeng. 2011; 108: 2261-2269. (C) 2011 Wiley Periodicals, Inc.