Xylo- and cello-oligosaccharide oxidation by gluco-oligosaccharide oxidase from Sarocladium strictum and variants with reduced substrate inhibition
1 Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada
2 Department of Biotechnology and Chemical Technology, Aalto University, Kemistintie 1 D1, Espoo 02150, Finland
3 Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 27, Helsinki 00014, Finland
Biotechnology for Biofuels 2013, 6:148 doi:10.1186/1754-6834-6-148Published: 12 October 2013
The oxidation of carbohydrates from lignocellulose can facilitate the synthesis of new biopolymers and biochemicals, and also reduce sugar metabolism by lignocellulolytic microorganisms, reserving aldonates for fermentation to biofuels. Although oxidoreductases that oxidize cellulosic hydrolysates have been well characterized, none have been reported to oxidize substituted or branched xylo-oligosaccharides. Moreover, this is the first report that identifies amino acid substitutions leading to GOOX variants with reduced substrate inhibition.
The recombinant wild type gluco-oligosaccharide oxidase (GOOX) from the fungus Sarocladium strictum, along with variants that were generated by site-directed mutagenesis, retained the FAD cofactor, and showed high activity on cello-oligosaccharide and xylo-oligosaccharides, including substituted and branched xylo-oligosaccharides. Mass spectrometric analyses confirmed that GOOX introduces one oxygen atom to oxidized products, and 1H NMR and tandem mass spectrometry analysis confirmed that oxidation was restricted to the anomeric carbon. The A38V mutation, which is close to a predicted divalent ion-binding site in the FAD-binding domain of GOOX but 30 Å away from the active site, significantly increased the kcat and catalytic efficiency of the enzyme on all oligosaccharides. Eight amino acid substitutions were separately introduced to the substrate-binding domain of GOOX-VN (at positions Y72, E247, W351, Q353 and Q384). In all cases, the Km of the enzyme variant was higher than that of GOOX, supporting the role of corresponding residues in substrate binding. Most notably, W351A increased Km values by up to two orders of magnitude while also increasing kcat up to 3-fold on cello- and xylo-oligosaccharides and showing no substrate inhibition.
This study provides further evidence that S. strictum GOOX has broader substrate specificity than the enzyme name implies, and that substrate inhibition can be reduced by removing aromatic side chains in the -2 binding subsite. Of the enzyme variants, W351A might be particularly advantageous when oxidizing oligosaccharides present at high substrate concentrations often experienced in industrial processes.