The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases

Abstract

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases,CfLPMO10 andTbLPMO10 fromCellulomonas fimiandThermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducingCtCBM3a, from theClostridium thermocellumcellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact ofCtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM fromCfLPMO10 or the introduction of a family 10 CBM fromCellvibrio japonicusLPMO10B intoTbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.

Keywords: carbohydrate binding modules; carbohydrate-binding protein; cellulases; enzyme; glycoside hydrolase; lignocellulose degradation; lytic polysaccharide monooxygenases; plant cell wall; protein engineering.

FIGURE 1.

HPAEC analysis of LPMOs. A and B, the HPAEC profiles of the reaction products for CfLPMO10 and CfLPMO10_CD (black and gray, respectively) (A) and for TbLPMO10 and TbLPMO10_CD (black and gray, respectively) (B). G3, G4, G5, and G6 are cellotriose, cellotetraose, cellopentaose, and cellohexaose, respectively. C, the gluconic acid produced by the full-length constructs and enzymes without CBMs on three different substrates. Oxidized (DPXox) and non-oxidized products (GX), where X is the degree of polymerization (DP), are indicated. Error bars indicate means ± S.E.

FIGURE 2.

MALDI-TOF analysis of products released by CtLPMO10 and TbLPMO10. A, the different oligosaccharides generated with DPs indicated. B, details of the cellohexaose species. GlcLA and GlcA represent the lactone and aldonic acid adducts, respectively. The blue bars in A correspond to the regions detailed in B. The asterisk indicates the peaks that are indicative of double C1+C4 oxidation, as they are 2 Da smaller than the corresponding C1-oxidized products.

FIGURE 3.

HPAEC analysis of products released by CfLPMO10 and TbLPMO10 to show evidence for C4 oxidation by the C. fimi enzyme.

FIGURE 4.

Fluorometric assay for the generation of hydrogen peroxide by variants of CfLPMO10. a.u., arbitrary units.

FIGURE 5.

Qualitative cellulose binding assays. A, B, and C, binding to Avicel (5% (w/v)), PASC (1% (w/v)), and BMCC (0.35% (w/v)), respectively. Lane a, starting material; lane b, non-bound material in the supernatant after the cellulose had been pelleted; lanes c and d, wash (lane c) and material eluted from washed and pelleted cellulose by boiling in 10% SDS (lane d). Experiments were carried out on ice using 80 μg of proteins in 200 μl of 20 mm sodium phosphate buffer, pH 8.0. 10 mm EDTA was added to the samples containing CfLPMO10_CD and TbLPMO10_CD to prevent any catalytic activity.

FIGURE 6.

Quantification of HPAEC analysis of limit products released from different forms of cellulose. G3, G4, G5, and G6 are cellotriose, cellotetraose, cellopentaose, and cellohexaose, respectively.

FIGURE 7.

Gluconic acid produced from different substrates. The chromatographs for each time point are shown in supplemental Fig. S1 and supplemental Fig. S2. Error bars indicate means ± S.E.

FIGURE 8.

Synergy between LPMOs and cellulases CjCel6A and CjCel5B. The data shown are for 100-h incubations of PASC and BMCC with the enzymes indicated. LPMOs and cellulases were used at 1 and 0.5 μm, respectively.