The 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. The 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. The 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.

Microbial bioconversion of photosynthetic biomass is a promising approach to the generation of biofuels and other bioproducts. However, rapid, high-yield, and simple processes are essential for successful applications. Here, biomass from the rapidly growing photosynthetic marine cyanobacterium Synechococcus sp. PCC 7002 was fermented using yeast into bioethanol.

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.

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.