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

Continuous recycling of enzymes during production of lignocellulosic bioethanol in demonstration scale

Recycling of enzymes in production of lignocellulosic bioethanol has been tried for more than 30 years. So far, the successes have been few and the experiments have been carried out at conditions far from those in an industrially feasible process. Here we have tested continuous enzyme recycling at demonstration scale using industrial process conditions (high dry matter content and low enzyme dosage) for a period of eight days. The experiment was performed at the Inbicon demonstration plant (Kalundborg, Denmark) capable of converting four tonnes of wheat straw per hour. 20% of the fermentation broth was recycled to the hydrolysis reactor while enzyme dosage was reduced by 5%. The results demonstrate that recycling enzymes by this method can reduce overall enzyme consumption and may also increase the ethanol concentrations in the fermentation broth. Our results further show that recycling fermentation broth also opens up the possibility of lowering the dry matter content in hydrolysis and fermentation while still maintaining high ethanol concentrations.M.O. Haven wishes to thank the Danish Agency for Science, Technology, and Innovation, grant no. 09-053694 for financial support. The other authors wish to thank the European Seventh Framework Program, grant no. 239379 (the KACELLE project) for financial support

Thermal unfolding and refolding of a lytic polysaccharide monooxygenase from <em>Thermoascus</em> aurantiacus

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes which promote the degradation of recalcitrant polysaccharides like cellulose or chitin. Here, we have investigated the thermostability of an LPMO from Thermoascus aurantiacus (TaLPMO9A). TaLPMO9A was found to retain most of its initial activity after incubating at 100 °C while its apparent melting temperature (Tm) is 69 °C at neutral pH. Interestingly, our studies show that holoTaLPMO9A, apoTaLPMO9A and deglycosylated TaLPMO9A can fold back to their original conformation upon lowering the temperature. In the presence of β-mercaptoethanol the protein does not refold. Activity of TaLPMO9A and refolded TaLPMO9A was studied by an Amplex® Red assay as well as by TaLPMO9A catalysed oxidation of phosphoric acid swollen cellulose (PASC). These studies confirm the functional regain of TaLPMO9A activity upon going through one cycle of unfolding and refolding. The thermal unfolding and refolding of TaLPMO9A was measured spectroscopically. Utilizing the two-state model, detailed thermodynamic parameters were obtained for holoTaLPMO. Furthermore, we have investigated the kinetics of TaLPMO9A unfolding and refolding. Our results have implications in understanding LPMO stability, which is crucial for the efficient application of LPMOs as biocatalysts during biomass degradation

Solvent consumption in non-catalytic alcohol solvolysis of biorefinery lignin

Identified solvent reaction routes in alcohol solvolysis of lignin where lignin-oil incorporation through covalent bonding is the only desired reaction.</p

On the roles of AA15 lytic polysaccharide monooxygenases derived from the termite Coptotermes gestroi

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which catalyze the oxidative cleavage of polysaccharides. LPMOs belonging to family 15 in the Auxiliary Activity (AA) class from the Carbohydrate-Active Enzyme database are found widespread across the Tree of Life, including viruses, algae, oomycetes and animals. Recently, two AA15s from the firebrat Thermobia domestica were reported to have oxidative activity, one towards cellulose or chitin and the other towards chitin, signalling that AA15 LPMOs from insects potentially have different biochemical functions. Herein, we report the identification and characterization of two family AA15 members from the lower termite Coptotermes gestroi. Addition of Cu(II) to CgAA15a or CgAA15b had a thermostabilizing effect on both. Using ascorbate and O2 as co-substrates, CgAA15a and CgAA15b were able to oxidize chitin, but showed no activity on celluloses, xylan, xyloglucan and starch. Structural models indicate that the LPMOs from C. gestroi (CgAA15a/CgAA15b) have a similar fold but exhibit key differences in the catalytic site residues when compared to the cellulose/chitin-active LPMO from T. domestica (TdAA15a), especially the presence of a non-coordinating phenylalanine nearby the Cu ion in CgAA15a/b, which appears as a tyrosine in the active site of TdAA15a. Despite the overall similarity in protein folds, however, mutation of the active site phenylalanine in CgAA15a to a tyrosine did not expanded the enzymatic specificity from chitin to cellulose. Our data show that CgAA15a/b enzymes are likely not involved in lignocellulose digestion but might play a role in termite developmental processes as well as on chitin and nitrogen metabolisms

How chip size impacts steam pretreatment effectiveness for biological conversion of poplar wood into fermentable sugars

BACKGROUND: Woody biomass is highly recalcitrant to enzymatic sugar release and often requires significant size reduction and severe pretreatments to achieve economically viable sugar yields in biological production of sustainable fuels and chemicals. However, because mechanical size reduction of woody biomass can consume significant amounts of energy, it is desirable to minimize size reduction and instead pretreat larger wood chips prior to biological conversion. To date, however, most laboratory research has been performed on materials that are significantly smaller than applicable in a commercial setting. As a result, there is a limited understanding of the effects that larger biomass particle size has on the effectiveness of steam explosion pretreatment and subsequent enzymatic hydrolysis of wood chips. RESULTS: To address these concerns, novel downscaled analysis and high throughput pretreatment and hydrolysis (HTPH) were applied to examine whether differences exist in the composition and digestibility within a single pretreated wood chip due to heterogeneous pretreatment across its thickness. Heat transfer modeling, Simons’ stain testing, magnetic resonance imaging (MRI), and scanning electron microscopy (SEM) were applied to probe the effects of pretreatment within and between pretreated wood samples to shed light on potential causes of variation, pointing to enzyme accessibility (i.e., pore size) distribution being a key factor dictating enzyme digestibility in these samples. Application of these techniques demonstrated that the effectiveness of pretreatment of Populus tremuloides can vary substantially over the chip thickness at short pretreatment times, resulting in spatial digestibility effects and overall lower sugar yields in subsequent enzymatic hydrolysis. CONCLUSIONS: These results indicate that rapid decompression pretreatments (e.g., steam explosion) that specifically alter accessibility at lower temperature conditions are well suited for larger wood chips due to the non-uniformity in temperature and digestibility profiles that can result from high temperature and short pretreatment times. Furthermore, this study also demonstrated that wood chips were hydrated primarily through the natural pore structure during pretreatment, suggesting that preserving the natural grain and transport systems in wood during storage and chipping processes could likely promote pretreatment efficacy and uniformity