Lytic polysaccharide monooxygenases utilise reducing agents within the biomass substrate to act synergistically with canonical hydrolases to enhance cellulose deconstruction.

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BackgroundCellulose amorphogenesis, described as the non-hydrolytic “opening up” or disruption of a cellulosic substrate, is becoming increasingly recognized as one of the key steps in the enzymatic deconstruction of cellulosic biomass when used as a feedstock for fuels and chemicals production. Although this process is thought to play a major role in facilitating hydrolysis, the lack of quantitative techniques capable of accurately describing the molecular-level changes occurring in the substrate during amorphogenesis has hindered our understanding of this process.ResultsIn this work, techniques for measuring changes in cellulose accessibility are reviewed and a new quantitative assay method is described. Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibres during amorphogenesis. The extents of phosphoric acid-induced and Swollenin-induced changes to cellulose accessibility were successfully quantified using this technique.ConclusionsThe adsorption of substructure-specific CBMs can be used to accurately quantify the extent of changes to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick, accurate and quantitative measure of the accessibility of cellulosic substrates. Expanding the range of CBMs used for adsorption studies to include those specific for such compounds as xylan or mannan should also allow for the accurate quantitative tracking of the accessibility of these and other polymers within the lignocellulosic biomass matrix.

A key limitation in the overall hydrolysis process is the restricted access that the hydrolytic enzymes have due to the macro-and-micro structure of cellulose and its association with hemicellulose and lignin. Previous work has shown that several non-hydrolytic proteins can disrupt cellulose structure and boost the activity of hydrolytic enzymes when purer forms of cellulose are used. In the work reported here, Swollenin primarily disrupted the hemicellulosic fraction of pretreated corn stover, resulting in the solubilisation of monomeric and oligomeric sugars. Although Swollenin showed little synergism when combined with the cellulase monocomponents exoglucanase (CEL7A) and endoglucanase (CEL5A), it showed pronounced synergism with xylanase monocomponents Xylanase GH10 and Xylanase GH11, resulting in the release of significantly more xylose (>300%). It appears that Swollenin plays a role in amorphogenesis and that its primary action is enhancing access to the hemicellulose fraction that limits or masks accessibility to the cellulose component of lignocellulosic substrates.

Effective enzymatic hydrolysis of insoluble cellulose requires the synergistic action of a suite of cellulase components. Most previous studies have only assessed cellulase synergism on model cellulosic substrates. When the actions of individual and combinations of cellulases (Cel7A, Cel6A, Cel7B, Cel5A) were assessed on various pretreated lignocellulosic substrates, Cel7A was shown to be the major contributor to overall cellulose hydrolysis, with the other enzymes synergistically enhancing its hydrolytic efficiency, at least partially, by facilitating Cel7A desorption (assessed by a double-sandwich enzyme-linked immunosorbent assay). When the influences of various substrate physicochemical characteristics on the effectiveness of enzyme synergism were assessed, a strong relationship was observed between cellulose accessibility (as determined by the cellulose binding module technique) and the degree of synergism, with greater synergy observed on the more disorganized/accessible cellulose surface.

Background: Fiber fragmentation is thought to occur at dislocations, which are potential targets for the non-hydrolytic protein, Swollenin. Results: Changes in cellulose morphology within dislocations were assessed using fluorescent CBMs; Swollenin appeared to promote fragmentation at these sites. Conclusion: Swollenin targets and disrupts cellulose at fiber dislocations. Significance: Fragmentation is a key step in cellulose deconstruction and is enhanced by the actions of Swollenin.

The underlying mechanisms of the recalcitrance of biomass to enzymatic deconstruction are still not fully understood, and this hampers the development of biomass based fuels and chemicals. With water being necessary for most biological processes, it is suggested that interactions between water and biomass may be key to understanding and controlling biomass recalcitrance. This study investigates the correlation between biomass recalcitrance and the constraint and retention of water by the biomass, using SO pretreated spruce, a common feedstock for lignocellulosic biofuel production, as a substrate to evaluate this relationship. The water retention value (WRV) of the pretreated materials was measured, and water constraint was assessed using time domain Low Field Nuclear Magnetic Resonance (LFNMR) relaxometry. WRV increased with pretreatment severity, correlating to reduced recalcitrance, as measured by hydrolysis of cellulose using commercial enzyme preparations. Water constraint increased with pretreatment severity, suggesting that a higher level of biomass-water interaction is indicative of reduced recalcitrance in pretreated materials. Both WRV and water constraint increased significantly with reductions in particle size when pretreated materials were further milled, suggesting that particle size plays an important role in biomass water interactions. It is suggested that WRV may be a simple and effective method for measuring and comparing biomass recalcitrance. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:146-153, 2017.

To enhance overall sugar recovery, poplar was steam pretreated in two stages. First, under mild pretreatment conditions (160 °C, 15 min) using various acid catalysts (H 2 SO 4 , SO 2 , oxalic, citric) to assess optimum hemicellulose recovery, followed by an uncatalyzed, second pretreatment (200 °C, 5 min), to facilitate enzymatic hydrolysis of the cellulose. The first stage solubilized and recovered about 50% of the xylan, as compared to the 30% obtained in the more severe, single-stage pretreatment. As surfactants are known to increase hemicellulose accessibility during pretreatments, lignosulfonates were added to birch xylan and poplar, increasing xylose yields and retaining 60 mmol/kg strong acid groups, even after the second pretreatment stage and extensive washing, suggesting that lignosulfonates adsorbed to the substrate and enhanced cellulose accessibility. This was confirmed by the increased water retention values and Direct Orange dye adsorption. A two-stage steam pretreatment, incorporating lignosulfonate addition, increased cellulose hydrolysis from 75 to 92%.