Cellotriose

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Synthetic polymers are resistant to biological attack, resulting in their long-term accumulation in landfills and in natural aquatic and terrestrial habitats. Lytic polysaccharide monooxygenases (LPMOs) are enzymes which oxidatively cleave the polysaccharide chains in recalcitrant polysaccharides such as cellulose. It has been widely hypothesised that LPMOs could be used to aid in the enzymatic breakdown of synthetic polymers. Herein, through the use of biochemical assays, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) we show that LPMOs can bind to polyethylene terephthalate (PET), and - in doing so - the hydrophobic surface of PET becomes more hydrophilic such that product release is boosted by subsequent treatment with classical PETases. The boosting effect is however, only observed in reactions when the LPMO and the PETase are added sequentially rather than simultaneously to the PET. Moreover, the same boosting effect is also seen when a catalytically-inactive mutant of LPMO is used, showing that the principal means by which AA9 LPMOs boost the degradation of synthetic polymers is through their role as a "hydrophobin" rather than as an oxygenase. Indeed, in accord with this role of LPMOs, we further show that this effect can be extended to other ostensibly 'non-catalytic' proteins beyond LPMOs, such as bovine serum albumin and lactate dehydrogenase.

Fluorescence labeling with N-(1-naphthyl)ethylenediamine is highly effective for quantifying oxidized reducing end groups (REGs) in cellulosic materials. When combined with size exclusion chromatography in DMAc/LiCl, along with fluorescence / multiple-angle laser light scattering / refractive index detection, a detailed profile of C1-oxidized REGs relative to the molecular weight distribution of the cellulosic material can be obtained. In this work, the derivatization process was extensively optimized, to be carried out heterogeneously in the solvent N-methyl-2-pyrrolidone. Furthermore, we show that to achieve high selectivity for carboxyl groups at the C1 position, keto and aldehyde groups need to be selectively reduced (e.g., by NaBH₄), and carboxyl groups other than at C1 need to be blocked (e.g., by methylation with (trimethylsilyl)diazomethane) prior to fluorescence labeling of carboxyl groups at C1 position. Finally, we demonstrate the practical value of the analytical method by measuring the content of the C1-oxidized REGs in cellulose samples after chemical (by Pinnick oxidation) or enzymatic (by treatment with C1-oxidizing LPMO enzymes) oxidation of various pulp samples.

The aim of the work was to compare the water retention and moisture sorption of viscose (CV) and cotton (Co) fibers carboxymethylated from aqueous media, in presence of NaOH, with sodium monochloroacetate. It was shown previously that under the same treatment conditions, the degree of carboxymethylation was higher in CV and so was the depth within fiber structures to which the carboxymethylation reactions occurred. It was also shown previously, that in terms of their capacity for sorption of a cationic dye (methylene blue), the Co performed better than CV. In this work, the same fibers were tested for their water retention and moisture sorption propensities. The two were sensitive both to the degree of carboxymethylation and the inherent properties of fibers (accessibility, degree of swelling, hornification). But the moisture sorption levels were less sensitive to the degree of carboxymethylation and more to inherent fiber properties whereas the reverse was observed for water retention. In contrast to the prior observations with dye sorption, CV performed better than Co in both moisture sorption and water retention. The poor performance of CV in dye sorption was attributed to the greater depth of carboxymethylation within the fibers that hindered dye permeation, but the same feature was observed to result in better performance (water retention) or not to hinder performance (moisture sorption). These observations highlight the contrasting effects that may arise, of a given set of treatment parameters (fiber type, alkali level in treatment), on efficacy of the product performance.

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, using a single copper cofactor bound in a conserved histidine brace with a more variable second coordination sphere. Cellulose-active LPMOs in the fungal AA9 family and in a subset of bacterial AA10 enzymes contain a His-Gln-Tyr second sphere motif, whereas other cellulose-active AA10s have an Arg-Glu-Phe motif. To shine a light on the impact of this variation, we generated single, double, and triple mutations changing the His²¹⁶–Gln²¹⁹-Tyr²²¹ motif in cellulose- and chitin-oxidizing MaAA10B toward Arg-Glu-Phe. These mutations generally reduced enzyme performance due to rapid inactivation under turnover conditions, showing that catalytic fine-tuning of the histidine brace is complex and that the roles of these second sphere residues are strongly interconnected. Studies of copper reactivity showed remarkable effects, such as an increase in oxidase activity following the Q219E mutation and a strong dependence of this effect on the presence of Tyr at position 221. In reductant-driven reactions, differences in oxidase activity, which lead to different levels of in situ generated H₂O₂, correlated with differences in polysaccharide-degrading ability. The single Q219E mutant displayed a marked increase in activity on chitin in both reductant-driven reactions and reactions fueled by exogenously added H₂O₂. Thus, it seems that the evolution of substrate specificity in LPMOs involves both the extended substrate-binding surface and the second coordination sphere.

Realising a fully circular bioeconomy requires the valorisation of lignocellulosic biomass. Cellulose is the most attractive component of lignocellulose but depolymerisation is inefficient, expensive and resource intensive requiring substantial volumes of potable water. Seawater is an attractive prospective replacement, however seawater tolerant enzymes are required for the development of seawater-based biorefineries. Here, we report a halophilic cellobiohydrolase SMECel6A, identified and isolated from a salt marsh meta-exo-proteome dataset with high sequence divergence to previously characterised cellobiohydrolases. SMECel6A contains a glycoside hydrolase family 6 (GH6) domain and a carbohydrate binding module family 2 (CBM2) domain. Characterisation of recombinant SMECel6A revealed SMECel6A to be active upon crystalline and amorphous cellulose. Mono- and oligosaccharide product profiles revealed cellobiose as the major hydrolysis product confirming SMECel6A as a cellobiohydrolase. We show SMECel6A to be halophilic with optimal activity achieved in 0.5X seawater displaying 80.6 ± 6.93% activity in 1 × seawater. Structural predictions revealed similarity to a characterised halophilic cellobiohydrolase despite sharing only 57% sequence identity. Sequential thermocycling revealed SMECel6A had the ability to partially reversibly denature exclusively in seawater retaining significant activity. Our study confirms that salt marsh ecosystems harbour enzymes with attractive traits with biotechnological potential for implementation in ionic solution based bioprocessing systems.

Penicillium roqueforti is used worldwide in the production of blue-veined cheese. The blue-green colour derives from pigmented spores formed by fungal growth. Using a combination of bioinformatics, targeted gene deletions, and heterologous gene expression we discovered that pigment formation was due to a DHN-melanin biosynthesis pathway. Systematic deletion of pathway genes altered the arising spore colour, yielding white to yellow-green to red-pink-brown phenotypes, demonstrating the potential to generate new coloured strains. There was no consistent impact on mycophenolic acid production as a result of pathway interruption although levels of roquefortine C were altered in some deletants. Importantly, levels of methyl-ketones associated with blue-cheese flavour were not impacted. UV-induced colour mutants, allowed in food production, were then generated. A range of colours were obtained and certain phenotypes were successfully mapped to pathway gene mutations. Selected colour mutants were subsequently used in cheese production and generated expected new colourations with no elevated mycotoxins, offering the exciting prospect of use in future cheese manufacture.

Lytic polysaccharide monooxygenases (LPMOs) are important for the effective depolymerization of recalcitrant polysaccharides. Despite their recognized importance, reported synergies with hydrolytic enzymes are often modest. We show that the kinetics of the LPMO-catalyzed depolymerization of chitin and cellulose are strongly affected by copper availability and the degree of enzyme copper saturation. Importantly, reactions with non-copper-saturated LPMOs are relatively slow but stable, which may be beneficial under industrial biomass processing conditions, whereas reactions with copper-saturated LPMOs are fast, but may lead to rapid enzyme inactivation. We show that this relates to the release of copper by damaged LPMOs that may be scavenged by apo-LPMOs, which then become activated. These effects of copper and copper saturation vary with the substrate concentration, which affects the rate of oxidative damage. We conclude that management of LPMO copper saturation and, perhaps, the use of copper chelators provides opportunities for optimizing the use of these powerful enzymes.

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C–H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H₂O₂ splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.

Background: The combination of cellulase and lytic polysaccharide monooxygenase (LPMO) is known to boost enzymatic saccharification of cellulose. Although the synergy between cellulases (GH5, 6 or 7) and LPMOs (AA9) has been extensively studied, the interplay between other glycoside hydrolase and LPMO families remains poorly understood. Results: In this study, two cellulolytic enzyme-encoding genes SmBglu12A and SmLpmo10A from Streptomyces megaspores were identified and heterologously expressed in Escherichia coli. The recombinant SmBglu12A is a non-typical endo-β-1,4-glucanase that preferentially hydrolyzed β-1,3-1,4-glucans and slightly hydrolyzed β-1,4-glucans and belongs to GH12 family. The recombinant SmLpmo10A belongs to a C1-oxidizing cellulose-active LPMO that catalyzed the oxidation of phosphoric acid swollen cellulose to produce celloaldonic acids. Moreover, individual SmBglu12A and SmLpmo10A were both active on barley β-1,3-1,4-glucan, lichenan, sodium carboxymethyl cellulose, phosphoric acid swollen cellulose, as well as Avicel. Furthermore, the combination of SmBglu12A and SmLpmo10A enhanced enzymatic saccharification of phosphoric acid swollen cellulose by improving the native and oxidized cello-oligosaccharides yields. Conclusions: These results proved for the first time that the AA10 LPMO was able to boost the catalytic efficiency of GH12 glycoside hydrolases on cellulosic substrates, providing another novel combination of glycoside hydrolase and LPMO for cellulose enzymatic saccharification.