Enhanced Polysaccharide Binding and Activity on Linear β-Glucans through Addition of Carbohydrate-Binding Modules to Either Terminus of a Glucooligosaccharide Oxidase.
The gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) is a single domain flavoenzyme that favourably oxidizes gluco- and xylo- oligosaccharides. In the present study, GOOX was shown to also oxidize plant polysaccharides, including cellulose, glucomannan, β-(1→3,1→4)-glucan, and xyloglucan, albeit to a lesser extent than oligomeric substrates. To improve GOOX activity on polymeric substrates, three carbohydrate binding modules (CBMs) from Clostridium thermocellum, namely CtCBM3 (type A), CtCBM11 (type B), and CtCBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-CtCBM3 and GOOX-CtCBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (kcat) on cellotetraose by up to 50%. All CBM fusions selectively enhanced GOOX binding to soluble and insoluble polysaccharides, and the immobilized enzyme on a solid cellulose surface remained stable and active. In addition, the CBM fusions increased the activity of GOOX on soluble glucomannan by up to 30% and on insoluble crystalline as well as amorphous cellulose by over 50%.
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
Maryam
M
Foumani
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
Thu V
TV
Vuong
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
Benjamin
B
MacCormick
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada.
Emma R
ER
Master
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D001203:Ascomycota / Q000201:enzymology*; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D004586:Electrophoresis; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism; D006207:Half-Life; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature; D013780:Tetroses / Q000378:metabolism; D047071:beta-Glucans / Q000378:metabolism*
D004800:Enzymes, Immobilized; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D013780:Tetroses; D047071:beta-Glucans; C048468:cellotetraose; D002482:Cellulose; D000429:Alcohol Oxidoreductases; C072250:glucooligosaccharide oxidase