Enhanced Polysaccharide Binding and Activity on Linear β-Glucans through Addition of Carbohydrate-Binding Modules to Either Terminus of a Glucooligosaccharide Oxidase

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%.

Fig 1. Schematic representation of wild-type GOOX and GOOX fusions.

Schematic representation of carbohydrate binding modules fused to the amino and carboxyl terminal end of glucooligosaccharide oxidase from Sarocladium strictum. Natural linkers were used in C-terminal fusions while TP linkers were used in N-terminal fusions.

Fig 2. Affinity gel electrophoresis (AGE) of wild-type GOOX and CBM fusions.

Purified proteins were subjected to AGE using a 7.5% (w/v) polyacrylamide gel containing A: no polysaccharides, B: β-glucan, C: xyloglucan, D: glucomannan, E: carboxymethyl cellulose. The final concentration of each polysaccharide was 0.01%. Bovine serum albumin (BSA) was used as a reference.

Fig 3. Activity of wild-type GOOX and CBM fusions on polysaccharides.

A: crystalline cellulose (Avicel, 0.5%), B: regenerated amorphous cellulose (RAC, 0.2%), and C: glucomannan from konjac (0.1%). Substrate concentrations were optimized to measure initial rates of reaction. One unit corresponds to 1 μmol of product per min. Error bars represents standard deviations; n = 3. The doted line represents the activity of wild-type GOOX.

Fig 4. Frequency—dissipation plot of wild-type GOOX and CtCBM3-GOOX binding to cellulose.

Changes in frequency (Δf) and dissipation (ΔD) during 1.5 μg/mL enzyme addition (1), 50 mM Tris-HCl pH 8 buffer washing (2) and 0.5 mM cellobiose addition (3) in the experiments with CtCBM3-GOOX (green, triangle) and wild-type GOOX (red, square). The total running time is 210 min. Linear fitting for cellulose binding of the CBM fusion (dashed arrow) and the wild-type (solid arrow) were analyzed by GraphPad Prism 5.