Investigating the role of AA9 LPMOs in enzymatic hydrolysis of differentially steam-pretreated spruce.

BACKGROUND: To realize the full potential of softwood-based forest biorefineries, the bottlenecks of enzymatic saccharification of softwood need to be better understood. Here, we investigated the potential of lytic polysaccharide monooxygenases (LPMO9s) in softwood saccharification. Norway spruce was steam-pretreated at three different severities, leading to varying hemicellulose retention, lignin condensation, and cellulose ultrastructure. Hydrolyzability of the three substrates was assessed after pretreatment and after an additional knife-milling step, comparing the efficiency of cellulolytic Celluclast + Novozym 188 and LPMO-containing Cellic CTec2 cocktails. The role of Thermoascus aurantiacus TaLPMO9 in saccharification was assessed through time-course analysis of sugar release and accumulation of oxidized sugars, as well as wide-angle X-ray scattering analysis of cellulose ultrastructural changes. RESULTS: Glucose yield was 6% (w/w) with the mildest pretreatment (steam pretreatment at 210 °C without catalyst) and 66% (w/w) with the harshest (steam pretreatment at 210 °C with 3%(w/w) SO2) when using Celluclast + Novozym 188. Surprisingly, the yield was lower with all substrates when Cellic CTec2 was used. Therefore, the conditions for optimal LPMO activity were tested and it was found that enough O2 was present over the headspace and that the reducing power of the lignin of all three substrates was sufficient for the LPMOs in Cellic CTec2 to be active. Supplementation of Celluclast + Novozym 188 with TaLPMO9 increased the conversion of glucan by 1.6-fold and xylan by 1.5-fold, which was evident primarily in the later stages of saccharification (24-72 h). Improved glucan conversion could be explained by drastically reduced cellulose crystallinity of spruce substrates upon TaLPMO9 supplementation. CONCLUSION: Our study demonstrated that LPMO addition to hydrolytic enzymes improves the release of glucose and xylose from steam-pretreated softwood substrates. Furthermore, softwood lignin provides enough reducing power for LPMOs, irrespective of pretreatment severity. These results provided new insights into the potential role of LPMOs in saccharification of industrially relevant softwood substrates.

Thermoresponsive Glycopolymers Based on Enzymatically Synthesized Oligo-ß-Mannosyl Ethyl Methacrylates and N-Isopropylacrylamide.

We present here a series of thermoresponsive glycopolymers in the form of poly(N-isopropylacrylamide)-co-(2-[ß-manno[oligo]syloxy] ethyl methacrylate)s. These copolymers were prepared from oligo-ß-mannosyl ethyl methacrylates that were synthesized through enzymatic catalysis, and were subsequently investigated with respect to their aggregation and phase behavior in aqueous solution using a combination of 1H NMR spectroscopy, dynamic light scattering, cryogenic transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS). The thermoresponsive glycopolymers were prepared by conventional free radical copolymerization of different mixtures of 2-(ß-manno[oligo]syloxy)ethyl methacrylates (with either one or two saccharide units) and N-isopropylacrylamide (NIPAm). The results showed that below the lower critical solution temperature (LCST) of poly(NIPAm), the glycopolymers readily aggregate into nanoscale structures, partly due to the presence of the saccharide moieties. Above the LCST of poly(NIPAm), the glycopolymers rearrange into a heterogeneous mixture of fractal and disc/globular aggregates. Cryo-TEM and SAXS data demonstrated that the presence of the pendant ß-mannosyl moieties in the glycopolymers induces a gradual conformational change over a wide temperature range. Even though the onset of this transition is not different from the LCST of poly(NIPAm), the gradual conformational change offers a variation of the temperature-dependent properties in comparison to poly(NIPAm), which displays a sharp coil-to-globule transition. Importantly, the compacted form of the glycopolymers shows a larger colloidal stability compared to the unmodified poly(NIPAm). In addition, the thermoresponsiveness can be conveniently tuned by varying the sugar unit-length and the oligo-ß-mannosyl ethyl methacrylate content.

Bicontinuous cubic liquid crystalline phase nanoparticles stabilized by softwood hemicellulose.

The colloidal stability of lipid based cubosomes, aqueous dispersion of inverse bicontinuous cubic phase, can be significantly increased by a stabilizer. The most commonly used stabilizers are non-ionic tri-block copolymers, poloxamers, which adsorb at the lipid-water interface and hence sterically stabilize the dispersion. One of the challenges with these synthetic polymers is the effect on the internal structure of the cubosomes and the potential toxicity when these nanoparticles are applied as nanomedicine platforms. The natural polysaccharide, softwood hemicellulose, has been proved to be an excellent stabilizer for oil-in-water emulsions, partially due to the presence of hydrophobic lignin in the extract which to some extent is associated to hemicellulose. Herein, we reported for the first time cubosomes stabilized by two types of softwood hemicelluloses, where one is extracted through thermomechanical pulping (TMP, low lignin content) and the other obtained from sodium-based sulfite liquor (SSL, high lignin content). The effect of the two hemicellulose samples on the colloidal stability and structure of monoolein-based cubosomes have been investigated via DLS, SAXS, AFM and cryo-TEM. The data obtained suggest that both types of the hemicelluloses stabilize monoolein (GMO) based cubosomes in water without significantly affecting their size, morphology and inner structure. SSL-extracted hemicellulose yields the most stable cubosomes, likely due to the higher content of lignin in comparison to TMP-stabilized ones. In addition, the stability of these particles was tested under physiological conditions relevant to possible application as drug carriers.

On the interaction of softwood hemicellulose with cellulose surfaces in relation to molecular structure and physicochemical properties of hemicellulose.

The substantial part of the water-soluble hemicellulose fraction, obtained when processing cellulose to produce paper and other products, has so far been discarded. The aim of this work is to reveal the interfacial properties of softwood hemicellulose (galactoglucomannan, GGM) in relation to their molecular and solution structure. In this study the sugar composition of GGM was characterised by chemical analysis as well as 1D and 2D NMR spectroscopy. Previously it has been demonstrated that hemicellulose has high affinity towards cellulose and has the ability to alter the properties of cellulose based products. This study is focused on the interactions between hemicellulose and the cellulose surface. Therefore, adsorption to hydrophobized silica and cellulose surfaces of two softwood hemicellulose samples and structurally similar seed hemicelluloses (galactomannans, GMs) was studied with ellipsometry, QCM-D and neutron reflectometry. Aqueous solutions of all samples were characterized with light scattering to determine how the degree of side-group substitution and molecular weight affect the conformation and aggregation of these polymers in the bulk. In addition, hemicellulose samples were studied with SAXS to investigate backbone flexibility. Light scattering results indicated that GM polymers form globular particles while GGMs were found to form rod-like aggregates in the solution. The polysaccharides exhibit higher adsorption to cellulose than on hydrophobic surfaces. A clear correlation between the increase in molecular weight of polysaccharides and increasing adsorbed amount on cellulose was observed, while the adsorbed amount on the hydrophobic surface was fairly independent of the molecular weight. The obtained layer thickness was compared with bulk scattering data and the results indicated flat conformation of the polysaccharides on the surface.

Facile control of surfactant lamellar phase transition and adsorption behavior.

This study sets out to investigate the effect of the presence of small water-soluble additives on the tunability of the surfactant gel-to-liquid crystalline (Lß-Lα) phase transition temperature (T m) for a bilayer-forming cationic surfactant and the phase behavior of such surfactant systems on dilution. This is strongly driven by the fact that this type of cationic surfactant has many interesting unanswered scientific questions and has found applications in various areas such as consumer care, the petrochemical industry, food science, etc. The underlying surfactant/additive interactions and the interfacial behavior of lamellar surfactant systems including the surfactant deposition on surfaces can provide new avenues to develop novel product formulations. We have examined dioctadecyldimethyl ammonium chloride (DODAC) in the presence of small polar additives, with respect to the phase behavior upon dilution and the deposition on silica. Differential scanning calorimetry (DSC) is used to track the transition temperature, T m, and synchrotron and laboratory-based small and wide-angle X-ray scattering (SAXS and WAXS) were used to determine the self-assembled surfactant structure below and above the T m. DSC scans showed that upon dilution the additives could be removed from the surfactant bilayer which in turn tuned the T m. A spontaneous transition from a liquid crystalline (Lα) phase to a gel (Lß) phase on dilution was demonstrated, which indicated that additives could be taken out from the Lα phase. By means of in situ null ellipsometry, the deposition of a diluted surfactant Lß phase upon replacement of bulk solution by deionized water was followed. This technique enables time-resolved monitoring of the deposited surfactant layer thickness and adsorbed amount, which allows us to understand the deposition on surfaces. Robust layers at least one bilayer-thick were deposited onto the surface and shown to be irreversibly adsorbed due to poor surfactant solvency in water. The thickest layer of surfactant deposited after dilution was found for mixtures with small amounts of additive since high amounts might lead to a phase-separated system.

ß-Mannanase-catalyzed synthesis of alkyl mannooligosides.

ß-Mannanases catalyze the conversion and modification of ß-mannans and may, in addition to hydrolysis, also be capable of transglycosylation which can result in enzymatic synthesis of novel glycoconjugates. Using alcohols as glycosyl acceptors (alcoholysis), ß-mannanases can potentially be used to synthesize alkyl glycosides, biodegradable surfactants, from renewable ß-mannans. In this paper, we investigate the synthesis of alkyl mannooligosides using glycoside hydrolase family 5 ß-mannanases from the fungi Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C). To evaluate ß-mannanase alcoholysis capacity, a novel mass spectrometry-based method was developed that allows for relative comparison of the formation of alcoholysis products using different enzymes or reaction conditions. Differences in alcoholysis capacity and potential secondary hydrolysis of alkyl mannooligosides were observed when comparing alcoholysis catalyzed by the three ß-mannanases using methanol or 1-hexanol as acceptor. Among the three ß-mannanases studied, TrMan5A was the most efficient in producing hexyl mannooligosides with 1-hexanol as acceptor. Hexyl mannooligosides were synthesized using TrMan5A and purified using high-performance liquid chromatography. The data suggests a high selectivity of TrMan5A for 1-hexanol as acceptor over water. The synthesized hexyl mannooligosides were structurally characterized using nuclear magnetic resonance, with results in agreement with their predicted ß-conformation. The surfactant properties of the synthesized hexyl mannooligosides were evaluated using tensiometry, showing that they have similar micelle-forming properties as commercially available hexyl glucosides. The present paper demonstrates the possibility of using ß-mannanases for alkyl glycoside synthesis and increases the potential utilization of renewable ß-mannans.