title: Fusion of a xylan-binding module to gluco-oligosaccharide oxidase increases activity and promotes stable immobilization.
abstract: The xylan-binding module Clostridium thermocellum CBM22A was successfully fused to a gluco-oligosaccharide oxidase, GOOX-VN, from Sarocladium strictum via a short TP linker, allowing the fused protein to effectively bind different xylans. The presence of the CtCBM22A at the N-terminal of GOOX-VN increased catalytic activity on mono- and oligo-saccharides by 2-3 fold while not affecting binding affinity to these substrates. Notably, both GOOX-VN and its CBM fusion also showed oxidation of xylo-oligosaccharides with degrees of polymerization greater than six. Whereas fusion to CtCBM22A did not alter the thermostability of GOOX-VN or reduce substrate inhibition, CtCBM22A_GOOX-VN could be immobilized to insoluble oat spelt xylan while retaining wild-type activity. QCM-D analysis showed that the fused enzyme remained bound during oxidation. These features could be harnessed to generate hemicellulose-based biosensors that detect and quantify the presence of different oligosaccharides.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Thu V
authors.initials: TV
authors.lastname: Vuong
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Emma R
authors.initials: ER
authors.lastname: Master
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000737:chemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014990:Xylans / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D014990:Xylans; C007916:hemicellulose; D000429:Alcohol Oxidoreductases; C072250:glucooligosaccharide oxidase
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