Fusion of a Xylan-Binding Module to Gluco-Oligosaccharide Oxidase Increases Activity and Promotes Stable Immobilization

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.

Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability

“…Fusing Ct CBM22A to Y300A also increased k cat values of the enzyme on all sugars, up to 67% in the case of cellotriose, from 670 min −1 to 1,120 min −1 (Table 1). A similar effect was previously seen when Ct CBM22A was fused to the N-terminus of the wild-type GOOX23. The K m values of Ct CBM22A_Y300A on monosaccharides including glucose and xylose were also decreased by around 45%, resulting in higher catalytic efficiency (Table 1).…”

“…As Ct CBM22A does not bind xylose and glucose27, the observed improvement in K m and catalysis on monosaccharides, as well as slight thermal activation at 40 °C (Fig. 2) by Ct CBM22A_Y300A, is likely due to a subtle structural change induced in the Y300A variant upon fusion to Ct CBM22A, as previously proposed for GOOX23.…”

“…Expression screening by immuno-colony blot, cell growth, protein expression and purification were conducted following previously reported methods23. Briefly, cells were grown at 15 °C and 250 rpm for 5 days, and 0.5% methanol was added every 24 h to induce recombinant protein expression.…”

“…The production of oxidized sugars was determined by continuously measuring the co-production of H 2 O 2 at 37 °C for 15 min using a chromogenic horseradish peroxidase assay23. Activity measurements were performed using 16 nM of each GOOX variant and 0.5 mM substrate; kinetic measurements were performed using the following substrate concentrations: 0.05–300 mM glucose; 0.05–1,200 mM xylose; 0.05–20 mM cellobiose; 0.01–10 mM cellotriose, xylobiose and xylotriose; 0.01–4 mM of longer cello- and xylo-oligosaccharides.…”

“…The HPAEC-PAD samples were eluted at 0.25 mL/min using sodium acetate gradient (0–1,000 mM) in 100 mM NaOH23. Chromatograms were viewed and analyzed using Chromeleon 7.2 (Dionex, USA).…”