Abstract
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 %.
Highlights
Oxidation of hydroxyl groups to carbonyls can enhance the gelation, thickening, and metal sequestration potential of polysaccharides [1] and be harnessed to modify corresponding surface properties through subsequent chemical derivatization [2]
When using the standard Pichia expression protocol to produce N-terminal carbohydrate binding modules (CBMs) fusions, degradation products that corresponded to the size of the wild type gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) and CBMs separately were observed by SDS-PAGE
Unchanged Km values confirmed that the fused CBMs did not compete with the GOOX subsites for oligosaccharide binding
Summary
Oxidation of hydroxyl groups to carbonyls can enhance the gelation, thickening, and metal sequestration potential of polysaccharides [1] and be harnessed to modify corresponding surface properties through subsequent chemical derivatization [2]. Pursuant to these objectives, chemicals such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), sodium periodate, and halide ions including I- and Br- have been used to respectively oxidize polysaccharides at primary hydroxyl positions, hydroxyl groups at positions C2 and C3, and hydroxyl groups at positions C1 and C6 [1].
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