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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Alcohol Oxidoreductases / chemistry*
-
Alcohol Oxidoreductases / genetics
-
Alcohol Oxidoreductases / metabolism*
-
Amino Acid Sequence
-
Clostridium thermocellum / enzymology
-
Clostridium thermocellum / genetics
-
Enzyme Stability
-
Enzymes, Immobilized / chemistry*
-
Enzymes, Immobilized / genetics
-
Enzymes, Immobilized / metabolism*
-
Models, Molecular
-
Molecular Sequence Data
-
Oxidation-Reduction
-
Polysaccharides / chemistry
-
Protein Conformation
-
Recombinant Fusion Proteins / chemistry*
-
Recombinant Fusion Proteins / genetics
-
Recombinant Fusion Proteins / metabolism*
-
Xylans / metabolism*
Substances
-
Enzymes, Immobilized
-
Polysaccharides
-
Recombinant Fusion Proteins
-
Xylans
-
hemicellulose
-
Alcohol Oxidoreductases
-
glucooligosaccharide oxidase
Grants and funding
This work was supported by the Government of Ontario (ORF-RE-05-005) and the Natural Sciences and Engineering Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.