David Cannella

Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content

BackgroundThe recent discovery of accessory proteins that boost cellulose hydrolysis has increased the economical and technical efficiency of processing cellulose to bioethanol. Oxidative enzymes (e.g. GH61) present in new commercial enzyme preparations have shown to increase cellulose conversion yields. When using pure cellulose substrates it has been determined that both oxidized and unoxidized cellodextrin products are formed. We report the effect of oxidative activity in a commercial enzyme mix (Cellic CTec2) upon overall hydrolysis, formation of oxidized products and impact on β-glucosidase activity. The experiments were done at high solids loadings using a lignocellulosic substrate simulating commercially relevant conditions.ResultsThe Cellic CTec2 contained oxidative enzymes which produce gluconic acid from lignocellulose. Both gluconic and cellobionic acid were produced during hydrolysis of pretreated wheat straw at 30% WIS. Up to 4% of released glucose was oxidized into gluconic acid using Cellic CTec2, whereas no oxidized products were detected when using an earlier cellulase preparation Celluclast/Novozym188. However, the cellulose conversion yield was 25% lower using Celluclast/Novozym188 compared to Cellic CTec2. Despite the advantage of the oxidative enzymes, it was shown that aldonic acids could be problematic to the hydrolytic enzymes. Hydrolysis experiments revealed that cellobionic acid was hydrolyzed by β-glucosidase at a rate almost 10-fold lower than for cellobiose, and the formed gluconic acid was an inhibitor of the β-glucosidase.Interestingly, the level of gluconic acid varied significantly with temperature. At 50°C (SHF conditions) 35% less gluconic acid was produced compared to at 33°C (SSF conditions). We also found that in the presence of lignin, no reducing agent was needed for the function of the oxidative enzymes.ConclusionsThe presence of oxidative enzymes in Cellic CTec2 led to the formation of cellobionic and gluconic acid during hydrolysis of pretreated wheat straw and filter paper. Gluconic acid was a stronger inhibitor of β-glucosidase than glucose. The formation of oxidized products decreased as the hydrolysis temperature was increased from 33° to 50°C. Despite end-product inhibition, the oxidative cleavage of the cellulose chains has a synergistic effect upon the overall hydrolysis of cellulose as the sugar yield increased compared to using an enzyme preparation without oxidative activity.

Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer

Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert.

Light-driven oxidation of polysaccharides by photosynthetic pigments and a metalloenzyme.

Oxidative processes are essential for the degradation of plant biomass. A class of powerful and widely distributed oxidative enzymes, the lytic polysaccharide monooxygenases (LPMOs), oxidize the most recalcitrant polysaccharides and require extracellular electron donors. Here we investigated the effect of using excited photosynthetic pigments as electron donors. LPMOs combined with pigments and reducing agents were exposed to light, which resulted in a never before seen 100-fold increase in catalytic activity. In addition, LPMO substrate specificity was broadened to include both cellulose and hemicellulose. LPMO enzymes and pigment derivatives common in the environment of plant-degrading organisms thus form a highly reactive and stable light-driven system increasing the turnover rate and versatility of LPMOs. This light-driven system may find applications in biotechnology and chemical processing.

Cellulase inhibition by high concentrations of monosaccharides.

Biological degradation of biomass on an industrial scale culminates in high concentrations of end products. It is known that the accumulation of glucose and cellobiose, end products of hydrolysis, inhibit cellulases and decrease glucose yields. Aside from these end products, however, other monosaccharides such as mannose and galactose (stereoisomers of glucose) decrease glucose yields as well. NMR relaxometry measurements showed direct correlations between the initial T2 of the liquid phase in which hydrolysis takes place and the total glucose production during cellulose hydrolysis, indicating that low free water availability contributes to cellulase inhibition. Of the hydrolytic enzymes involved, those acting on the cellulose substrate, that is, exo- and endoglucanases, were the most inhibited. The β-glucosidases were shown to be less sensitive to high monosaccharide concentrations except glucose. Protein adsorption studies showed that this inhibition effect was most likely due to catalytic, and not binding, inhibition of the cellulases.

Lignocellulose pretreatment technologies affect the level of enzymatic cellulose oxidation by LPMO

Sugarcane bagasse, corn stover, and wheat straw are among the most available resources for the production of cellulosic ethanol. For these biomasses we study the influence of pre-treatment methods on the chemical composition, as well as on the subsequent reactions of enzymatic hydrolysis and oxidation of cellulose. The applied pre-treatment methods are organosolv, hydrothermal, and alkaline. Hydrothermally pretreated wheat straw gave the highest cellulose conversion with 80% glucose yield and 0.8% oxidized cellulose products. Recent studies have shown that lignin is able to boost the activity of the cellulose oxidizing enzyme lytic polysaccharide monooxygenase (LPMO). The highest activity of LPMO was observed for the hydrothermally pretreated biomasses, which also contained the highest level of lignin. All hydrolyses were done at high dry matter levels using a commercial enzyme preparation containing hydrolytic and oxidative enzymes.

Influence of high gravity process conditions on the environmental impact of ethanol production from wheat straw

Biofuel production processes at high gravity are currently under development. Most of these processes however use sugars or first generation feedstocks as substrate. This paper presents the results of a life cycle assessment (LCA) of the production of bio-ethanol at high gravity conditions from a second generation feedstock, namely, wheat straw. The LCA used lab results of a set of 36 process configurations in which dry matter content, enzyme preparation and loading, and process strategy were varied. The LCA results show that higher dry matter content leads to a higher environmental impact of the ethanol production, but this can be compensated by reducing the impact of enzyme production and use, and by polyethylene glycol addition at high dry matter content. The results also show that the renewable and non-renewable energy use resulting from the different process configurations ultimately determine their environmental impact.

On the formation and role of reactive oxygen species in light-driven LPMO oxidation of phosphoric acid swollen cellulose

Light-driven activation of lytic polysaccharide monooxygenases (LPMOs) has been attributed to the transfer of high redox potential electrons from excited photopigments to the enzyme. However, due to the formation of reactive oxygen species (ROS) in such a system, not only electrons from the pigments but also ROS could be part of the enzyme mechanism. This work investigates the role of ROS in the oxidation of phosphoric acid swollen cellulose (PASC) by a light-driven LPMO system. Our results clearly show that the addition of superoxide dismutase or catalase to remove ROS did not attenuate the capacity of the light-driven LPMO system to oxidize PASC, as measured by formation of oxidized oligosaccharides. We conclude that ROS are not part of the light-driven LPMO activation; hence, transfer of high redox potential electrons from the excited photopigment to the LPMO remains the most likely mechanism under the conditions tested in this study.

Laccase-derived lignin compounds boost cellulose oxidative enzymes AA9.

BackgroundThe discovery of lignin as activator for the redox enzyme lytic polysaccharide monooxygenases (LPMOs) for the oxidation of cell-wall polysaccharides opens a new scenario for investigation of the interplay between different lignocellulose-degrading enzymes. The lignin-active enzymes in one hand, and the carbohydrate active in the other, are linked through a variety of electrons carrier molecules either derived from lignin or enzymatically transferred. Likewise, in nature, many lignocellulose-degrading organisms are expressing those enzymes simultaneously, and we wanted to test if a major commercial available lignin oxidase enzyme, i.e., laccase could benefit and synergize the activity of the LPMOs by depolymerizing the insoluble lignin.ResultsIn this work, two fungal laccases together with a mediator (ABTS) were used to isolate low-molecular-weight lignin from lignocellulosic biomass. The isolated lignins were used as electron donors for activation of LPMOs. A direct correlation between the low-molecular-weight lignin isolated with laccases and an increased activity of a cellulolytic cocktail containing LPMO was found when pure cellulose was hydrolyzed. We then tried to implement existing commercial cellulases cocktail with laccase enzymes, but under the conditions tested, the co-incubation of laccases with LPMOs showed a substrate competition towards oxygen inhibiting the LPMO. In addition, we found that laccase treatment may cause other modifications to pure cellulose, rendering the material more recalcitrant for enzymatic saccharification.ConclusionsLaccase-mediated system was able to depolymerize lignin from pre-treated and native sugarcane bagasse and wheat straw, and the released phenolic molecules were able to donate electrons to LPMO enzymes boosting the overall enzymatic hydrolysis of cellulose. Likewise, other poly-phenol oxidase, we might have just started showing possible pros or cons in applying several oxidase enzymes for a simultaneous degradation of cellulose and lignin, and we found that the competition towards oxygen and their different consumption rates must be taken into account for any possible co-application.

Light-Induced Electron Transfer Protocol for Enzymatic Oxidation of Polysaccharides

Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes that oxidize the most recalcitrant polysaccharides and require extracellular electron donors. The role of electron donation to redox enzymes is pivotal since a nonefficient electron transfer might result in partial activity or reduced kinetics. In this protocol we show the effect of using excited photosynthetic pigments combined with reducing agents as efficient electron donors for monooxygenases. The light-induced electron transfer can enhance the oxidation ability of LPMOs up to ten times.