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

<div><p>The xylan-binding module <i>Clostridium thermocellum</i> CBM22A was successfully fused to a gluco-oligosaccharide oxidase, GOOX-VN, from <i>Sarocladium strictum</i> via a short TP linker, allowing the fused protein to effectively bind different xylans. The presence of the <i>Ct</i>CBM22A 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 <i>Ct</i>CBM22A did not alter the thermostability of GOOX-VN or reduce substrate inhibition, <i>Ct</i>CBM22A_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.</p></div

Substrate inhibition and thermostability of GOOX-VN and <i>Ct</i>CBM22A_GOOX-VN.

<p>(A): The activity of 16 nM of each enzyme on different concentrations of cellotriose, up to 10 mM; (B): Enzymes were incubated for 1 hr at temperatures from 30 to 60°C, and then the residual activities of 16 nM enzyme on 0.5 mM cellobiose were measured.</p

HPAEC-PAD separation of native and oxidized xylo-oligosaccharides.

<p>(A): Xylo-oligosaccharide standard from xylose to xylohexaose (X1 to X6, respectively) before oxidation (top) and after the oxidation of <i>Ct</i>CBM22A_GOOX-VN, which released oxidized xylose to xylohexaose (ox.X1 to ox.X6, respectively) (bottom). (B): A chromatogram of soluble oat spelt xylan without any enzyme treatment. (C): Xylanase treatment of soluble oat spelt xylan (black solid), GOOX-VN treated soluble oat spelt xylan (red dashed) and <i>Ct</i>CBM22A_GOOX-VN treated soluble oat spelt xylan (green dotted dashed); peaks formed by oxidation were asterisked.</p

Binding of GOOX-VN and its CBM fusion on soluble polymeric substrates.

<p>(A): Control, no polymeric substrate; (B): 0.01% soluble oat spelt xylan; (C): 0.01% beechwood xylan; (D): 0.01% propoxylated wheat bran hemicellulose. Lanes 1 and 2: BSA as the negative control; lanes 3 and 4: GOOX-VN; lanes 5 and 6: <i>Ct</i>CBM22A_GOOX-VN.</p

QCM-D responses on binding and activity of the CBM fusion.

<p>(A): After the frequency and dissipation responses (n = 5) of xylan-coated sensors were stable in the reaction buffer (50 mM Tris-HCl pH 8.0), the sensors were exposed to 10 µg/mL <i>Ct</i>CBM22A_GOOX-VN, followed again with the reaction buffer, and then 0.5 mM cellobiose. (B): The thickness of the adsorbed layers was calculated using the Voigt model provided in the Q-Tools program (Q-sense, Sweden). The thickness values should be considered on a relative basis and should not be considered absolute values as the fitting includes approximations of layer density properties <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095170#pone.0095170-Eronen1" target="_blank">[30]</a>.</p

Structural model of the fusion protein.

<p>GOOX-VN was fused to <i>C. thermocellum</i> CBM22A (PDB ID: 1DYO) via a TP-rich linker. The model of GOOX-VN was built from the X-ray structure of GOOX-T1 (PDB ID: 2AXR) while the non-helical model of the linker was built from the X-ray structure of a <i>Bacillus subtilis</i> polysaccharide deacetylase (PDB ID: 1NY1). Xylohexaose (X6) was docked into the active site of GOOX-VN using Autodock 4. Top: the substrate-entrance view of the fused protein model; Bottom: the side view of the fused protein model.</p

Stability of the immobilized CBM fusion at room temperature.

<p><i>Ct</i>CBM22A_GOOX-VN was kept at room temperature with all reaction components, and then its activity on 0.5 mM cellobiose was measured every 24 hr. Relative activity (%) of the immobilized enzyme to the free form in the same assay conditions was shown.</p

Activity of GOOX-VN and <i>Ct</i>CBM22A_GOOOX-VN on hemicelluloses.

<p>^*Equal molar equivalents were used. 32 nM of each enzyme was assayed with 0.1% substrate.</p><p>– No activity was detected.</p

Grundlagen bibliometrischer Analysen

A chemo-enzymatic pathway was developed to transform 4-<i>O</i>-methyl­glucuronic acid (MeGlc<i>p</i>A) containing xylo-oligosaccharides from beechwood into clickable monomers capable of polymerizing at room temperature and in aqueous conditions to form unique poly­triazoles. While the gluco-oligosaccharide oxidase (GOOX) from <i>Sarocladium strictum</i> was used to oxidize C6-propargylated oligosaccharides, the acid–amine coupling reagents 1-ethyl-3-(3-(dimethylamino)­propyl) carbodiimide (EDAC) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) were employed and compared for their ability to append click functionalities to carboxylic acid groups of enzyme-treated oligosaccharides. While DMT-MM was a superior coupling reagent for this application, a triazine side product was observed during C-1 amidation. Resulting bifunctional xylo-oligosaccharide monomers were polymerized using a Cu­(I) catalyst, forming a soft gel which was characterized by ¹H NMR, confirming the triazole product

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

<div><p>The gluco-oligosaccharide oxidase from <i>Sarocladium strictum</i> 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 <i>Clostridium thermocellum</i>, namely <i>Ct</i>CBM3 (type A), <i>Ct</i>CBM11 (type B), and C<i>t</i>CBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-<i>Ct</i>CBM3 and GOOX-<i>Ct</i>CBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (<i>k</i>cat) 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 %.</p></div