H2 O2 feeding enables LPMO-assisted cellulose saccharification during simultaneous fermentative production of lactic acid.

Simultaneous saccharification and fermentation (SSF) is a well-known strategy for valorization of lignocellulosic biomass. Because the fermentation process typically is anaerobic, oxidative enzymes found in modern commercial cellulase cocktails, such as lytic polysaccharide monooxygenases (LPMOs), may be inhibited, limiting the overall efficiency of the enzymatic saccharification. Recent discoveries, however, have shown that LPMOs are active under anoxic conditions if they are provided with H2 O2 at low concentrations. In this study, we build on this concept and investigate the potential of using externally added H2 O2 to sustain oxidative cellulose depolymerization by LPMOs during an SSF process for lactic acid production. The results of bioreactor experiments with 100 g/L cellulose clearly show that continuous addition of small amounts of H2 O2 (at a rate of 80 µM/h) during SSF enables LPMO activity and improves lactic acid production. While further process optimization is needed, the present proof-of-concept results show that modern LPMO-containing cellulase cocktails such as Cellic CTec2 can be used in SSF setups, without sacrificing the LPMO activity in these cocktails.

Effects of Yeast Species and Processing on Intestinal Health and Transcriptomic Profiles of Atlantic Salmon (Salmo salar) Fed Soybean Meal-Based Diets in Seawater.

The objective of the current study was to examine the effects of yeasts on intestinal health and transcriptomic profiles from the distal intestine and spleen tissue of Atlantic salmon fed SBM-based diets in seawater. Cyberlindnera jadinii (CJ) and Wickerhamomyces anomalus (WA) yeasts were heat-inactivated with spray-drying (ICJ and IWA) or autolyzed at 50 °C for 16 h (ACJ and AWA), followed by spray-drying. Six diets were formulated, one based on fishmeal (FM), a challenging diet with 30% soybean meal (SBM) and four other diets containing 30% SBM and 10% of each of the four yeast fractions (i.e., ICJ, ACJ, IWA and AWA). The inclusion of CJ yeasts reduced the loss of enterocyte supranuclear vacuolization and reduced the population of CD8α labeled cells present in the lamina propria of fish fed the SBM diet. The CJ yeasts controlled the inflammatory responses of fish fed SBM through up-regulation of pathways related to wound healing and taurine metabolism. The WA yeasts dampened the inflammatory profile of fish fed SBM through down-regulation of pathways related to toll-like receptor signaling, C-lectin receptor, cytokine receptor and signal transduction. This study suggests that the yeast species, Cyberlindnera jadinii and Wickerhamomyces anomalus are novel high-quality protein sources with health-beneficial effects in terms of reducing inflammation associated with feeding plant-based diets to Atlantic salmon.

Alginate Degradation: Insights Obtained through Characterization of a Thermophilic Exolytic Alginate Lyase.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to polysaccharide lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics data set. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.2.2.26), which can degrade alginate, poly-ß-d-mannuronate, and poly-α-l-guluronate within a broad range of pHs, temperatures, and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as a catalytic acid, indeed is essential for catalysis, whereas mutation of tyrosine Y446, previously proposed to act as a catalytic base, did not affect enzyme activity. The observed reaction products are protonated and deprotonated forms of the 4,5-unsaturated uronic acid monomer, Δ, two hydrates of DEH (4-deoxy-l-erythro-5-hexulosuronate), which are formed after ring opening, and, finally, two epimers of a 5-member hemiketal called 4-deoxy-d-manno-hexulofuranosidonate (DHF), formed through intramolecular cyclization of hydrated DEH. The detection and nuclear magnetic resonance (NMR) assignment of these hemiketals refine our current understanding of alginate degradation.IMPORTANCE The potential markets for seaweed-derived products and seaweed processing technologies are growing, yet commercial enzyme cocktails for complete conversion of seaweed to fermentable sugars are not available. Such an enzyme cocktail would require the catalytic properties of a variety of different enzymes, where fucoidanases, laminarinases, and cellulases together with endo- and exo-acting alginate lyases would be the key enzymes. Here, we present an exo-acting alginate lyase that efficiently produces monomeric sugars from alginate. Since it is only the second characterized exo-acting alginate lyase capable of degrading alginate at a high industrially relevant temperature (≥60°C), this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation revealed previously undescribed rearrangement products of the unsaturated monomeric sugars generated from exo-acting lyases. The insight provided by the NMR assignment of these products facilitates future assessment of product formation by alginate lyases.

Production and characterization of yeasts grown on media composed of spruce-derived sugars and protein hydrolysates from chicken by-products.

BACKGROUND: A possible future shortage of feed protein will force mankind to explore alternative protein sources that can replace conventional soymeal or fishmeal. Several large industrial organic side-streams could potentially be upgraded to feed protein using a fermentation process to generate single cell protein. Yeast is the most widely accepted microorganism for production of single cell protein, because of its superior nutritional quality and acceptability among consumers. Here, we have assessed the growth of four different yeasts, Cyberlindnera jadinii, Wickerhamomyces anomalus, Blastobotrys adeninivorans and Thermosacc® Dry (Saccharomyces cerevisiae), on media composed of enzymatically saccharified sulfite-pulped spruce wood and hydrolysates of by-products from chicken, and we have characterized the resulting yeast biomass. RESULTS: Generally, the yeast grew very well on the spruce- and chicken-based medium, with typical yields amounting to 0.4-0.5 g of cell dry weight and 0.2-0.3 g of protein per g of sugar. B. adeninivorans stood out as the most versatile yeast in terms of nutrient consumption and in this case yields were as high as 0.9 g cells and 0.5 g protein per g of sugar. The next best performing yeast in terms of yield was W. anomalus with up to 0.6 g cells and 0.3 g protein per g sugar. Comparative compositional analyses of the yeasts revealed favorable amino acid profiles that were similar to the profiles of soymeal, and even more so, fish meal, especially for essential amino acids. CONCLUSIONS: The efficient conversion of industrial biomass streams to yeast biomass demonstrated in this study opens new avenues towards better valorization of these streams and development of sustainable feed ingredients. Furthermore, we conclude that production of W. anomalus or B. adeninivorans on this promising renewable medium may be potentially more efficient than production of the well-known feed ingredient C. jadinii. Further research should focus on medium optimization, development of semi-continuous and continues fermentation protocols and exploration of downstream processing methods that are beneficial for the nutritional values of the yeast for animal feed.

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives.

Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies and different enzyme process setups and present an in-depth discussion of the various enzyme types that are currently in use. We pay ample attention to the role of the recently discovered lytic polysaccharide monooxygenases (LPMOs), which have led to renewed interest in the role of redox enzyme systems in lignocellulose processing. Better understanding of the interplay between the various enzyme types, as they may occur in a commercial enzyme cocktail, is likely key to further process improvements.

Spruce sugars and poultry hydrolysate as growth medium in repeated fed-batch fermentation processes for production of yeast biomass.

The production of microbial protein in the form of yeast grown on lignocellulosic sugars and nitrogen-rich industrial residues is an attractive approach for reducing dependency on animal and plant protein. Growth media composed of enzymatically saccharified sulfite-pulped spruce wood, enzymatic hydrolysates of poultry by-products and urea were used for the production of single-cell protein. Strains of three different yeast species, Cyberlindnera jadinii, Wickerhamomyces anomalus and Blastobotrys adeninivorans, were cultivated aerobically using repeated fed-batch fermentation up to 25 L scale. Wickerhamomyces anomalus was the most efficient yeast with yields of 0.6 g of cell dry weight and 0.3 g of protein per gram of glucose, with cell and protein productivities of 3.92 g/L/h and 1.87 g/L/h, respectively. Using the conditions developed here for producing W. anomalus, it would take 25 industrial (200 m3) continuously operated fermenters to replace 10% of the fish feed protein used in Norway.

Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2.

Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) play an important role in the conversion of recalcitrant polysaccharides, but their mode of action has remained largely enigmatic. It is generally believed that catalysis by LPMOs requires molecular oxygen and a reductant that delivers two electrons per catalytic cycle. Using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show here that H2O2, rather than O2, is the preferred co-substrate of LPMOs. By controlling H2O2 supply, stable reaction kinetics are achieved, the LPMOs work in the absence of O2, and the reductant is consumed in priming rather than in stoichiometric amounts. The use of H2O2 by a monocopper enzyme that is otherwise cofactor-free offers new perspectives regarding the mode of action of copper enzymes. Furthermore, these findings have implications for the enzymatic conversion of biomass in Nature and in industrial biorefining.

Effects of post anaerobic digestion thermal hydrolysis on dewaterability and moisture distribution in digestates.

Organic waste fractions such as sewage sludge, food waste and manure can be stabilized by anaerobic digestion (AD) to produce renewable energy in the form of biogas. Following AD, the digested solid fraction (digestate) is usually dewatered to reduce the volume before transportation. Post-AD treatments such as the Post-AD thermal hydrolysis process (Post-AD THP) have been developed to improve the dewatering, but the mode of action is not well understood. In this study, samples from 32 commercial full-scale plants were used to assess the impact of Post-AD THP on a broad range of raw materials. Maximum dewatered cake solids after Post-AD THP was predicted by thermogravimetric analysis (TGA). Post-AD THP changed the moisture distribution of the samples by increasing the free water fraction. A consistent improvement in predicted dewatered cake solids was achieved across the 32 samples tested, on average increasing the dry solids concentration by 87%. A full-scale trial showed that dewatering Post-AD THP digestate at 80 °C improved dewatered cake solids above the predictions by TGA at 35 °C. In conclusion, dewatered cake solids were significantly improved by Post-AD THP, reducing the volume of dewatered cake for disposal.

Use of high metal-containing biogas digestates in cereal production - Mobility of chromium and aluminium.

Biogas digestate use as organic fertilizer has been widely promoted in recent years as a part of the global agenda on recycling waste and new sustainable energy production. Although many studies have confirmed positive effects of digestates on soil fertility, there is still lack of information on the potential adverse effects of digestates on natural soil heavy metal content, metal leaching and leaching of other pollutants. We have investigated the release of aluminium (Al) and chromium (Cr) from different soils treated with commercial digestates high in mentioned potentially problematic metals in a field experiment, while a greenhouse and a laboratory column experiment were used to address mobility of these metals in two other scenarios. Results obtained from the field experiment showed an increase in total concentrations for both investigated metals on plots treated with digestates as well as a significant increase of water-soluble Al concentrations. Factors that were found to be mostly affecting the metal mobility were dissolved organic carbon (DOC), pH and type of soil. Metal binding and free metal concentrations were modelled using the WHAM 7.0 software. Results indicated that the use of digestates with high metal content are comparable to use of animal manure with respect to metal leaching. Data obtained through chemical modelling for the samples from the field experiment suggested that an environmental risk from higher metal mobility has to be considered for Al. In the greenhouse experiment, measured concentrations of leached Cr at the end of the growing season were low for all treatments, while the concentration of leached Al from digestates was higher. The high irrigation column leaching experiment showed an increased leaching rate of Cr with addition of digestates.

Quantitative Metaproteomics Highlight the Metabolic Contributions of Uncultured Phylotypes in a Thermophilic Anaerobic Digester.

In this study, we used multiple meta-omic approaches to characterize the microbial community and the active metabolic pathways of a stable industrial biogas reactor with food waste as the dominant feedstock, operating at thermophilic temperatures (60°C) and elevated levels of free ammonia (367 mg/liter NH3-N). The microbial community was strongly dominated (76% of all 16S rRNA amplicon sequences) by populations closely related to the proteolytic bacterium Coprothermobacter proteolyticus. Multiple Coprothermobacter-affiliated strains were detected, introducing an additional level of complexity seldom explored in biogas studies. Genome reconstructions provided metabolic insight into the microbes that performed biomass deconstruction and fermentation, including the deeply branching phyla Dictyoglomi and Planctomycetes and the candidate phylum "Atribacteria" These biomass degraders were complemented by a synergistic network of microorganisms that convert key fermentation intermediates (fatty acids) via syntrophic interactions with hydrogenotrophic methanogens to ultimately produce methane. Interpretation of the proteomics data also suggested activity of a Methanosaeta phylotype acclimatized to high ammonia levels. In particular, we report multiple novel phylotypes proposed as syntrophic acetate oxidizers, which also exert expression of enzymes needed for both the Wood-Ljungdahl pathway and ß-oxidation of fatty acids to acetyl coenzyme A. Such an arrangement differs from known syntrophic oxidizing bacteria and presents an interesting hypothesis for future studies. Collectively, these findings provide increased insight into active metabolic roles of uncultured phylotypes and presents new synergistic relationships, both of which may contribute to the stability of the biogas reactor. IMPORTANCE: Biogas production through anaerobic digestion of organic waste provides an attractive source of renewable energy and a sustainable waste management strategy. A comprehensive understanding of the microbial community that drives anaerobic digesters is essential to ensure stable and efficient energy production. Here, we characterize the intricate microbial networks and metabolic pathways in a thermophilic biogas reactor. We discuss the impact of frequently encountered microbial populations as well as the metabolism of newly discovered novel phylotypes that seem to play distinct roles within key microbial stages of anaerobic digestion in this stable high-temperature system. In particular, we draft a metabolic scenario whereby multiple uncultured syntrophic acetate-oxidizing bacteria are capable of syntrophically oxidizing acetate as well as longer-chain fatty acids (via the ß-oxidation and Wood-Ljundahl pathways) to hydrogen and carbon dioxide, which methanogens subsequently convert to methane.

Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation.

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures.

A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides.

Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61-3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end.

The effect of storage conditions on microbial community composition and biomethane potential in a biogas starter culture.

A new biogas process is initiated by adding a microbial community, typically in the form of a sample collected from a functional biogas plant. This inoculum has considerable impact on the initial performance of a biogas reactor, affecting parameters such as stability, biogas production yields and the overall efficiency of the anaerobic digestion process. In this study, we have analyzed changes in the microbial composition and performance of an inoculum during storage using barcoded pyrosequencing of bacterial and archaeal 16S ribosomal RNA (rRNA) genes, and determination of the biomethane potential, respectively. The inoculum was stored at room temperature, 4 and -20 °C for up to 11 months and cellulose was used as a standard substrate to test the biomethane potential. Storage up to 1 month resulted in similar final methane yields, but the rate of methane production was reduced by storage at -20 °C. Longer storage times resulted in reduced methane yields and slower production kinetics for all storage conditions, with room temperature and frozen samples consistently giving the best and worst performance, respectively. Both storage time and temperature affected the microbial community composition and methanogenic activity. In particular, fluctuations in the relative abundance of Bacteroidetes were observed. Interestingly, a shift from hydrogenotrophic methanogens to methanogens with the capacity to perform acetoclastic methanogensis was observed upon prolonged storage. In conclusion, this study suggests that biogas inocula may be stored up to 1 month with low loss of methanogenic activity, and identifies bacterial and archaeal species that are affected by the storage.

Crystal structure and computational characterization of the lytic polysaccharide monooxygenase GH61D from the Basidiomycota fungus Phanerochaete chrysosporium.

Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain.

Improving nutrient fixation and dry matter content of an ammonium-rich anaerobic digestion effluent by struvite formation and clay adsorption.

The anaerobic digestion (AD) of organic wastes that contain nitrogen leads to its mineralization, yielding a digestate rich in ammonium (NH(4)(+)), an important fertilizing nutrient. The applicability of AD digestate as fertilizer can be improved by fixating the nutrients and increasing its dry matter content. Methods for the fixation and recovery of the digestate's NH(4)(+) and possible also PO(4)(3-) include struvite precipitation and adsorption in clay materials such as bentonite. These techniques were tested in batch experiments employing the liquid fraction of a digestate originating from the AD of a substrate mix containing lignocellulose, cattle manure and fish industrial waste. The concentration of NH(4)(+)-N in this digestate was 2,300 mg L⁻¹. Struvite precipitation conditions at a molar ratio of 1.2:1:1 (Mg²âº:NH(4)(+):PO(4)(3-)) and pH 9.5 were best in terms of simultaneous removal of NH(4)(+)-N (88%), PO(4)(3-) (60%) and soluble chemical oxygen demand (44%). Bentonite adsorption gave comparably high removal levels for NH(4)(+)-N (82%) and PO(4)(3-) (52%). Analysis of the precipitates' morphology and elemental composition confirmed their struvite and bentonite nature. Dry matter content was increased from 5.8% in the AD digestate to 27% and 22% in the struvite and bentonite sludges, respectively.

Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities.

BACKGROUND: The polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear. RESULTS: An LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid). CONCLUSIONS: This study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

Hallmarks of processivity in glycoside hydrolases from crystallographic and computational studies of the Serratia marcescens chitinases.

Degradation of recalcitrant polysaccharides in nature is typically accomplished by mixtures of processive and nonprocessive glycoside hydrolases (GHs), which exhibit synergistic activity wherein nonprocessive enzymes provide new sites for productive attachment of processive enzymes. GH processivity is typically attributed to active site geometry, but previous work has demonstrated that processivity can be tuned by point mutations or removal of single loops. To gain additional insights into the differences between processive and nonprocessive enzymes that give rise to their synergistic activities, this study reports the crystal structure of the catalytic domain of the GH family 18 nonprocessive endochitinase, ChiC, from Serratia marcescens. This completes the structural characterization of the co-evolved chitinolytic enzymes from this bacterium and enables structural analysis of their complementary functions. The ChiC catalytic module reveals a shallow substrate-binding cleft that lacks aromatic residues vital for processivity, a calcium-binding site not previously seen in GH18 chitinases, and, importantly, a displaced catalytic acid (Glu-141), suggesting flexibility in the catalytic center. Molecular dynamics simulations of two processive chitinases (ChiA and ChiB), the ChiC catalytic module, and an endochitinase from Lactococcus lactis show that the nonprocessive enzymes have more flexible catalytic machineries and that their bound ligands are more solvated and flexible. These three features, which relate to the more dynamic on-off ligand binding processes associated with nonprocessive action, correlate to experimentally measured differences in processivity of the S. marcescens chitinases. These newly defined hallmarks thus appear to be key dynamic metrics in determining processivity in GH enzymes complementing structural insights.

Lytic polysaccharide monooxygenase activity increases productive binding capacity of cellobiohydrolases on cellulose.

Cellobiohydrolases are crucial for cellulose breakdown, but their efficiency on crystalline cellulose is hampered by limited access to single chain ends to initiate hydrolysis. As a result, they depend on enzymes like lytic polysaccharide monooxygenases (LPMOs), which directly target the crystalline cellulose surface. This study investigated how LPMO pretreatment affected the productive binding capacity of a Trichoderma longibrachiatum cellobiohydrolase, TlCBHI, on crystalline cellulose by applying an amperometric cellobiose dehydrogenase biosensor. After the 24-hour of LPMO pretreatment, the productive binding capacity of TlCBHI significantly increased in all reactions. However, with a shorter 5-hour LPMO pretreatment, minimal to no effect on productive binding capacity was observed. Of note, all LPMO reactions were inactivated around this time point. This delayed LPMO effect suggests that the improved binding capacity for cellulases does not directly result from cellulose chain cleavage by LPMOs but rather from the cellulose decrystallization following the oxidative cleavage.

Engineering cellulases for conversion of lignocellulosic biomass.

Lignocellulosic biomass is a renewable source of energy, chemicals and materials. Many applications of this resource require the depolymerization of one or more of its polymeric constituents. Efficient enzymatic depolymerization of cellulose to glucose by cellulases and accessory enzymes such as lytic polysaccharide monooxygenases is a prerequisite for economically viable exploitation of this biomass. Microbes produce a remarkably diverse range of cellulases, which consist of glycoside hydrolase (GH) catalytic domains and, although not in all cases, substrate-binding carbohydrate-binding modules (CBMs). As enzymes are a considerable cost factor, there is great interest in finding or engineering improved and robust cellulases, with higher activity and stability, easy expression, and minimal product inhibition. This review addresses relevant engineering targets for cellulases, discusses a few notable cellulase engineering studies of the past decades and provides an overview of recent work in the field.

Visible light-exposed lignin facilitates cellulose solubilization by lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of crystalline polysaccharides such as cellulose and are crucial for the conversion of plant biomass in Nature and in industrial applications. Sunlight promotes microbial conversion of plant litter; this effect has been attributed to photochemical degradation of lignin, a major redox-active component of secondary plant cell walls that limits enzyme access to the cell wall carbohydrates. Here, we show that exposing lignin to visible light facilitates cellulose solubilization by promoting formation of H2O2 that fuels LPMO catalysis. Light-driven H2O2 formation is accompanied by oxidation of ring-conjugated olefins in the lignin, while LPMO-catalyzed oxidation of phenolic hydroxyls leads to the required priming reduction of the enzyme. The discovery that light-driven abiotic reactions in Nature can fuel H2O2-dependent redox enzymes involved in deconstructing lignocellulose may offer opportunities for bioprocessing and provides an enzymatic explanation for the known effect of visible light on biomass conversion.