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Altered substrate specificity of the gluco-oligosaccharide oxidase from Acremonium strictum
Maryam Foumani1, Thu V Vuong, Emma R Master
1Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, Canada M5S 3E5; telephone: +1-416-946-7861; fax: +1-416-978-8605.
Abstract:
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 kcat 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.
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