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.
Highlights
The Carbohydrate Active enZyme database (CAZy, http://www.cazy.org) has categorized carbohydrate-oxidizing enzymes into 11 auxiliary activity (AA) families based on sequence similarities
Protein fusion construction and production Both termini of GOOX-VN orient in the same direction (Fig. 1); while the C-terminus is positioned behind the active site, the N-terminus is positioned on the same planar surface as the substrate binding site
Designs for the fusion protein linked the CBM22A and the TP-rich linker to the N-terminus of GOOX-VN (Fig. 1)
Summary
The Carbohydrate Active enZyme database (CAZy, http://www.cazy.org) has categorized carbohydrate-oxidizing enzymes into 11 auxiliary activity (AA) families based on sequence similarities. While many carbohydrate oxidases have been reported to work on mono- or di-saccharides, the members of families AA3_1 [3], AA5_2 [4] and AA7 [5] show oxidation of larger oligosaccharides and even polysaccharides Among those oligosaccharide oxidases are gluco-oligosaccharide oxidases (GOOX, AA7, EC 1.1.3.x), from Sarocladium strictum strain CBS 346.70 and strain T1, which have been previously characterized by our group [6,7] and others [8,9,10], respectively. Among the GOOX variants from S. strictum CBS 346.70 is GOOX-VN [6]; the substrate specificity of GOOX-VN is not different from the recombinant wild-type enzyme [7] and the biochemical properties of GOOX-VN have been well characterized [6,7]
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