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.

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.

While it has been known for decades that enzymatic oxidation of lignin by laccases and peroxidases plays a role in microbial biomass conversion of lignin, it has only very recently become apparent that oxidative processes also play a major role in the conversion of polysaccharides. The latter process is carried out by so-called Lytic Polysaccharide MonoOxygenases (LPMOs) 1 , which are copper-dependent enzymes capable of breaking glycosidic bonds in polysaccharides, such as cellulose, xyloglucan, glucomannan, xylan, starch and chitin 2-9 . LPMO activity depends on the presence of molecular oxygen and requires an electron donor. So far, it has been shown that electrons can be provided by cellobiose dehydrogenase (CDH) 10 or by small molecule electron donors such as ascorbic acid 7 or gallic acid 6 . However, virtually nothing is known about how the LPMO-catalyzed redox reactions and electron transfers function within the plant cell wall matrix during biological decay.During biomass conversion by fungi, many of the lignin-and carbohydrate-active redox enzymes are expressed simultaneously with hydrolytic enzymes 11,12 , which points to a possible interplay between these enzyme systems. LPMO-encoding genes are abundant in the genomes of biomass degrading and plant pathogenic fungi, and when grown on lignocellulosic material, LPMOs are among the most highly expressed proteins [13][14][15][16] . Notably, there is ample evidence that LPMOs enhance the power of the fungal degradative enzyme machinery 17,18 . Today, LPMOs are important components in industrial enzyme cocktails used for saccharification of cellulosic biofuel feedstocks 19,20 .LPMOs are copper enzymes 6 , which cycle between Cu (I) and Cu (II) to activate molecular oxygen. Kim et al. (2014) suggested a mechanism involving the formation of a copper-oxyl radical that abstracts a hydrogen and then hydroxylates the substrate via an oxygen-rebound mechanism 21 . The details of oxygen activation were further elaborated by X-ray absorption studies of the active site copper, leading to the conclusion that the initial oxygen species is a super oxide 22 . During in vivo conditions, the electron donor may be CDH, but microorganisms may also utilize other approaches for providing electron donors to oxidative reactions. Lignin is one of the main structural components in plants and has an electron configuration that provides a low barrier for electron transfer. There are indications that lignin may act as electron donor for LPMOs 18,23 , but substantial evidence is scarce.One-electron transfer from lignin, proceeding via an outer sphere mechanism 24 , is well known and has been described for lignin oxidizing enzymes such as laccases 25 . The transfer may take place through direct interactions

Production of ethanol from lignocellulosic materials has a promising market potential, but the process is still only at pilot/demonstration scale due to the technical and economical difficulties of the process. Operating the process at very high solids concentrations (above 20% dry matter-DM) has proven essential for economic feasibility at industrial scale. Historically, simultaneous saccharification and fermentation (SSF) was found to give better ethanol yields compared to separate hydrolysis and fermentation (SHF), but data in literature are typically based on operating the process at low dry matter conditions. In this work the impact of selected enzyme preparation and processing strategy (SHF, presaccharification and simultaneous saccharification and fermentation-PSSF, and SSF) on final ethanol yield and overall performance was investigated with pretreated wheat straw up to 30% DM. The experiments revealed that an SSF strategy was indeed better than SHF when applying an older generation enzyme cocktail (Celluclast-Novozym 188). In case of the newer product Cellic CTec 2, SHF resulted in 20% higher final ethanol yield compared to SSF. It was possible to close the mass balance around cellulose to around 94%, revealing that the most relevant products could be accounted for. One observation was the presence of oxidized sugar (gluconic acid) upon enzymatic hydrolysis with the latest enzyme preparation. Experiments showed gluconic acid formation by recently discovered enzymatic class of lytic polysaccharides monoxygenases (LPMO's) to be depending on the processing strategy. The lowest concentration was achieved in SSF, which could be correlated with less available oxygen due to simultaneous oxygen consumption by the yeast. Quantity of glycerol and cell mass was also depending on the selected processing strategy.

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.