Assessing the role of redox partners in TthLPMO9G and its mutants: focus on H2O2 production and interaction with cellulose.

BACKGROUND: The field of enzymology has been profoundly transformed by the discovery of lytic polysaccharide monooxygenases (LPMOs). LPMOs hold a unique role in the natural breakdown of recalcitrant polymers like cellulose and chitin. They are characterized by a "histidine brace" in their active site, known to operate via an O2/H2O2 mechanism and require an electron source for catalytic activity. Although significant research has been conducted in the field, the relationship between these enzymes, their electron donors, and H2O2 production remains complex and multifaceted. RESULTS: This study examines TthLPMO9G activity, focusing on its interactions with various electron donors, H2O2, and cellulose substrate interactions. Moreover, the introduction of catalase effectively eliminates H2O2 interference, enabling an accurate evaluation of each donor's efficacy based on electron delivery to the LPMO active site. The introduction of catalase enhances TthLPMO9G's catalytic efficiency, leading to increased cellulose oxidation. The current study provides deeper insights into specific point mutations, illuminating the crucial role of the second coordination sphere histidine at position 140. Significantly, the H140A mutation not only impacted the enzyme's ability to oxidize cellulose, but also altered its interaction with H2O2. This change was manifested in the observed decrease in both oxidase and peroxidase activities. Furthermore, the S28A substitution, selected for potential engagement within the His1-electron donor-cellulose interaction triad, displayed electron donor-dependent alterations in cellulose product patterns. CONCLUSION: The interaction of an LPMO with H2O2, electron donors, and cellulose substrate, alongside the impact of catalase, offers deep insights into the intricate interactions occurring at the molecular level within the enzyme. Through rational alterations and substitutions that affect both the first and second coordination spheres of the active site, this study illuminates the enzyme's function. These insights enhance our understanding of the enzyme's mechanisms, providing valuable guidance for future research and potential applications in enzymology and biochemistry.

Two-Step Upcycling Process of Lignocellulose into Edible Bacterial Nanocellulose with Black Raspberry Extract as an Active Ingredient.

(1) Background: Bacterial nanocellulose (BNC) has gained in popularity over the years due to its outstanding properties such as renewability, biocompatibility, and bioavailability, and its use as an eco-friendly material of the future for replacing petrochemical products. (2) Methods: This research refers to the utilization of lignocellulose coming from wood waste via enzymatic hydrolysis to produce biopolymer BNC with an accumulation rate of 0.09 mg/mL/day. Besides its significant contribution to the sustainability, circularity, and valorization of biomass products, the obtained BNC was functionalized through the adsorption of black raspberry extract (BR) by simple soaking. (3) Results: BR contained 77.25 ± 0.23 mg GAE/g of total phenolics and 27.42 ± 0.32 mg CGE/g of total anthocyanins. The antioxidant and antimicrobial activity of BR was evaluated by DPPH (60.51 ± 0.18 µg/mL) and FRAP (1.66 ± 0.03 mmol Fe2+/g) and using a standard disc diffusion assay, respectively. The successful synthesis and interactions between BNC and BR were confirmed by FTIR analysis, while the morphology of the new nutrient-enriched material was investigated by SEM analysis. Moreover, the in vitro release kinetics of a main active compound (cyanidin-3-O-rutinoside) was tested in different release media. (4) Conclusions: The upcycling process of lignocellulose into enriched BNC has been demonstrated. All findings emphasize the potential of BNC-BR as a sustainable food industry material.

Evaluation of Growth and Production of High-Value-Added Metabolites in Scenedesmus quadricauda and Chlorella vulgaris Grown on Crude Glycerol under Heterotrophic and Mixotrophic Conditions Using Monochromatic Light-Emitting Diodes (LEDs).

This study aimed to examine the impact of crude glycerol as the main carbon source on the growth, cell morphology, and production of high-value-added metabolites of two microalgal species, namely Chlorella vulgaris and Scenedesmus quadricauda, under heterotrophic and mixotrophic conditions, using monochromatic illumination from light-emitting diodes (LEDs) emitting blue, red, yellow, and white (control) light. The findings indicated that both microalgae strains exhibited higher biomass yield on the mixotrophic growth system when compared to the heterotrophic one, while S. quadricauda generally performed better than C. vulgaris. In mixotrophic mode, the use of different monochromatic illumination affected biomass production differently on both strains. In S. quadricauda, growth rate was higher under red light (µmax = 0.89 d-1), while the highest biomass concentration and yield per gram of consumed glycerol were achieved under yellow light, reaching 1.86 g/L and Yx/s = 0.18, respectively. On the other hand, C. vulgaris demonstrated a higher growth rate on blue light (µmax = 0.45 d-1) and a higher biomass production on white (control) lighting (1.34 g/L). Regarding the production of metabolites, higher yields were achieved during mixotrophic mode in both strains. In C. vulgaris, the highest lipid (26.5% of dry cell weight), protein (63%), and carbohydrate (20.3%) contents were obtained under blue, red, and yellow light, respectively, thus indicating that different light wavelengths probably activate different metabolic pathways. Similar results were obtained for S. quadricauda with red light leading to higher lipid content, while white lighting caused higher production of proteins and carbohydrates. Overall, the study demonstrated the potential of utilizing crude glycerol as a carbon source for the growth and metabolite production of microalgae and, furthermore, revealed that the strains' behavior varied depending on lighting conditions.

Lytic polysaccharide monooxygenases as powerful tools in enzymatically assisted preparation of nano-scaled cellulose from lignocellulose: A review.

Nanocellulose, either in the form of fibers or crystals, constitutes a renewable, biobased, biocompatible material with advantageous mechanical properties that can be isolated from lignocellulosic biomass. Enzyme-assisted isolation of nanocellulose is an attractive, environmentally friendly approach that leads to products of higher quality compared to their chemically prepared counterparts. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively cleave the ß-1,4-glycosidic bond of polysaccharides upon activation of O2 or H2O2 and presence of an electron donor. Their use for treatment of cellulose fibers towards the preparation of nano-scaled cellulose is related to the ability of LPMOs to create nicking points on the fiber surface, thus facilitating fiber disruption and separation. The aim of this review is to describe the mode of action of LPMOs on cellulose fibers towards the isolation of nanostructures, thus highlighting their great potential for the production of nanocellulose as a novel value added product from lignocellulose.

Recent advances on key enzymatic activities for the utilisation of lignocellulosic biomass.

The field of enzymatic degradation of lignocellulose is actively growing and the recent updates of the last few years indicate that there is still much to learn. The growing number of protein sequences with unknown function in microbial genomes indicates that there is still much to learn on the mechanisms of lignocellulose degradation. In this review, a summary of the progress in the field is presented, including recent discoveries on the nature of the structural polysaccharides, new technologies for the discovery and functional annotation of gene sequences including omics technologies, and the novel lignocellulose-acting enzymes described. Novel enzymatic activities and enzyme families as well as accessory enzymes and their synergistic relationships regarding biomass breakdown are described. Moreover, it is shown that all the valuable knowledge of the enzymatic decomposition of plant biomass polymers can be employed towards the decomposition and upgrading of synthetic polymers, such as plastics.

Efficient production of nutraceuticals and lactic acid from lignocellulosic biomass by combining organosolv fractionation with enzymatic/fermentative routes.

The aim of this work was to investigate the use of isobutanol as organic solvent for the efficient delignification and fractionation of beechwood through the OxiOrganosolv process in the absence of any catalyst. The results demonstrate that cellulose-rich solid pulp produced after pretreatment is a source of fermentable sugars that can be easily hydrolyzed and serve as a carbon source in microbial fermentations for the production of omega-3 fatty acids and D-lactic acid. The C5 sugars are recovered in the aqueous liquid fractions and comprise a fraction rich in xylo-oligosaccharides with prebiotic potential. The maximum production of optically pure D-lactic from Lactobacillus delbrueckii sp. bulgaricus reached 51.6 g/L (0.57 g/gbiomass), following a simultaneous saccharification and fermentation strategy. Crypthecodenium cohnii accumulated up to 52.1 wt% lipids with a DHA content of 54.1 %, while up to 43.3 % hemicellulose recovery in form of oligosaccharides was achieved in the liquid fraction.

Conversion of organosolv pretreated hardwood biomass into 5-hydroxymethylfurfural (HMF) by combining enzymatic hydrolysis and isomerization with homogeneous catalysis.

BACKGROUND: Over the last few years, valorization of lignocellulosic biomass has been expanded beyond the production of second-generation biofuels to the synthesis of numerous platform chemicals to be used instead of their fossil-based counterparts. One such well-researched example is 5-hydroxymethylfurfural (HMF), which is preferably produced by the dehydration of fructose. Fructose is obtained by the isomerization of glucose, which in turn is derived by the hydrolysis of cellulose. However, to avoid harsh reaction conditions with high environmental impact, an isomerization step towards fructose is necessary, as fructose can be directly dehydrated to HMF under mild conditions. This work presents an optimized process to produce fructose from beechwood biomass hydrolysate and subsequently convert it to HMF by employing homogeneous catalysis. RESULTS: The optimal saccharification conditions were identified at 10% wt. solids loading and 15 mg enzyme/gsolids, as determined from preliminary trials on pure cellulose (Avicel® PH-101). Furthermore, since high rate glucose isomerization to fructose requires the addition of sodium tetraborate, the optimum borate to glucose molar ratio was determined to 0.28 and was used in all experiments. Among 20 beechwood solid pulps obtained from different organosolv pretreatment conditions tested, the highest fructose production was obtained with acetone (160 °C, 120 min), reaching 56.8 g/100 g pretreated biomass. A scale-up hydrolysis in high solids (25% wt.) was then conducted. The hydrolysate was subjected to isomerization eventually leading to a high-fructose solution (104.5 g/L). Dehydration of fructose to HMF was tested with 5 different catalysts (HCl, H3PO4, formic acid, maleic acid and H-mordenite). Formic acid was found to be the best one displaying 79.9% sugars conversion with an HMF yield and selectivity of 44.6% and 55.8%, respectively. CONCLUSIONS: Overall, this work shows the feasibility of coupling bio- and chemo-catalytic processes to produce HMF from lignocellulose in an environmentally friendly manner. Further work for the deployment of biocatalysts for the oxidation of HMF to its derivatives could pave the way for the emergence of an integrated process to effectively produce biobased monomers from lignocellulose.

Isolation and modification of nano-scale cellulose from organosolv-treated birch through the synergistic activity of LPMO and endoglucanases.

Nanocellulose isolation from lignocellulose is a tedious and expensive process with high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been cost-effective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (ζ-potential -19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (To = 301 °C, Tmax = 337 °C). Nanocelluloses were subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content.

Valorization of outer tunic of the marine filter feeder Ciona intestinalis towards the production of second-generation biofuel and prebiotic oligosaccharides.

BACKGROUND: One of the sustainable development goals focuses on the biomass-based production as a replacement for fossil-based commodities. A novel feedstock with vast potentials is tunicate biomass, which can be pretreated and fermented in a similar way to lignocellulose. Ciona intestinalis is a marine filter feeder that is cultivated to produce fish feed. While the inner tissue body is used for feed production, the surrounding tunic remains as a cellulose-rich by-product, which can be further separated into outer and inner tunic. Ethanol production from organosolv-pretreated whole-tunic biomass was recently validated. The aim of the present study was to evaluate the potential of organosolv pretreated outer-tunic biomass for the production of biofuels and cellobiose that is a disaccharide with prebiotic potential. RESULTS: As a result, 41.4 g/L of ethanol by Saccharomyces cerevisiae, corresponding to a 90.2% theoretical yield, was achieved under the optimal conditions when the tunicate biomass was pretreated at 195 °C for 60 min at a liquid-to-solid ratio of 50. In addition, cellobiose production by enzymatic hydrolysis of the pretreated tunicate biomass was demonstrated with a maximum conversion yield of 49.7 wt. %. CONCLUSIONS: The utilisation of tunicate biomass offers an eco-friendly and sustainable alternative for value-added biofuels and chemicals. The cultivation of tunicate biomass in shallow coastal sea improves the quality of the water and ensures sustainable production of fish feed. Moreover, there is no competition for arable land, which leaves the latter available for food and feed production.

Screening of Recombinant Lignocellulolytic Enzymes Through Rapid Plate Assays.

In the search for novel biomass-degrading enzymes through mining microbial genomes, it is necessary to apply functional tests during high-throughput screenings, which are capable of detecting enzymatic activities directly by way of plate assay. Using the most efficient expression systems of Escherichia coli and Pichia pastoris, the production of a high amount of His-tagged recombinant proteins could be thrived, allowing the one-step isolation by affinity chromatography. Here, we describe simple and efficient assay techniques for the detection of various biomass-degrading enzymatic activities on agar plates, such as cellulolytic, hemicellulolytic, and ligninolytic activities and their isolation using immobilized-metal affinity chromatography.

Valorization of exhausted olive pomace for the production of omega-3 fatty acids by Crypthecodinium cohnii.

Exhausted olive pomace (EOP) represents a potential candidate side stream to be utilized in biotechnological processes. EOP composition includes significant amounts of extractives and pectin, which are both usually discarded and are not utilized in the valorization process of the raw material. In this study, organosolv technology was optimized to remove the extractives and pectin using a Central Composite Rotatable Design. Optimal pretreatment conditions were predicted to be at 97.95 °C for 23.18 min, upon addition of 50% (v/v) EtOH in H2O, with 0.5% (w/v) of H2SO4 as catalyst. The composition analysis of liquid fraction revealed a high content of total sugars (17.58 g/L), galacturonic acid (7.05 g/L) and phenolic compounds (2.97 g/L). The liquid fraction was utilized as a carbon source by the heterotrophic marine microalgae Crypthecodinium cohnii, where it was shown to promote lipid accumulation up to 38.5% wt. of cell biomass, even without any additional detoxification step. This study is the first report that shows the use of galacturonic acid as carbon source for the growth of C. cohnii, while underpinning the use of EOP as a promising substrate for the development of zero-waste bioprocesses.

OxiOrganosolv: A novel acid free oxidative organosolv fractionation for lignocellulose fine sugar streams.

The valorization of lignocellulosic biomass towards the production of value-added products requires an efficient pretreatment/fractionation step. In this work we present a novel, acid-free, mildly oxidative organosolv delignification process -OxiOrganosolv- which employs oxygen gas to depolymerize and remove lignin. The results demonstrate that the OxiOrganosolv process achieved lignin removal as high as 97% in a single stage, with a variety of solvents; it was also efficient in delignifying both beechwood (hardwood) and pine (softwood), a task in which organosolv pretreatments have failed in the past. Minimal amounts of sugar degradation products were detected, while cellulose recovery was ~100% in the solid pulp. Enzymatic hydrolysis of pulps showed >80 wt% cellulose conversion to glucose. Overall, the OxiOrganosolv pretreatment has significant advantages, including high delignification efficiency of hardwood and softwood biomass, absence of acid homogeneous catalysis and all corresponding challenges involved, and close to zero losses of sugars to degradation products.

Utilization of lignocellulosic biomass towards the production of omega-3 fatty acids by the heterotrophic marine microalga Crypthecodinium cohnii.

Omega-3 fatty acids have become a commodity of high nutritional and commercial value; intensive fishing and its environmental and social cost has led researchers to seeking alternative more sustainable ways of producing them. Heterotrophic microalgae such as Crypthecodinium cohnii, a marine dinoflagellate, have the ability to utilize various substrates and accumulate high amounts of docosahexaenoic acid (DHA). In this work, a mild oxidative organosolv pretreatment of beechwood pulps was employed that allowed up to 95% of lignin removal in a single stage, thus yielding a cellulose-rich solid fraction. The enzymatic hydrolysates were evaluated for their ability to support the growth and lipid accumulation of C. cohnii in batch and fed-batch cultures; the results verified the successful microalgae growth, while DHA reached up to 43.5% of the cell's total lipids. The proposed bioprocess demonstrated the utilization of non-edible biomass towards high added value food supplements in a sustainable and efficient manner.

Valorization of waste forest biomass toward the production of cello-oligosaccharides with potential prebiotic activity by utilizing customized enzyme cocktails.

BACKGROUND: Production of value-added materials from lignocellulosic biomass residues is an emerging sector that has attracted much attention as it offers numerous benefits from an environmental and economical point of view. Non-digestible oligosaccharides represent a group of carbohydrates that are resistant to gastrointestinal digestion, and therefore, they are considered as potential prebiotic candidates. Such oligosaccharides can derive from the biomass cellulose fraction through a controlled enzymatic hydrolysis that eliminates the yield of monomers. RESULTS: In the present study, hydrolysis of organosolv-pretreated forest residues (birch and spruce) was tested in the presence of four cellulases (EG5, CBH7, CBH6, EG7) and one accessory enzyme (LPMO). The optimal enzyme combinations were comprised of 20% EG5, 43% CBH7, 22% TtLPMO, 10% PaCbh6a and 5% EG7 in the case of birch and 35% EG5, 45% CBH7, 10% TtLPMO, 10% PaCbh6a and 5% EG7 in the case of spruce, leading to 22.3% and 19.1 wt% cellulose conversion into cellobiose, respectively. Enzymatic hydrolysis was applied on scale-up reactions, and the produced oligosaccharides (consisted of > 90% cellobiose) were recovered and separated from glucose through nanofiltration at optimized temperature (50 °C) and pressure (10 bar) conditions, yielding a final product with cellobiose-to-glucose ratio of 21.1 (birch) and 20.2 (spruce). Cellobiose-rich hydrolysates were tested as fermentative substrates for different lactic acid bacteria. It was shown that they can efficiently stimulate the growth of two Lactobacilli strains. CONCLUSIONS: Controlled enzymatic hydrolysis with processive cellulases, combined with product recovery and purification, as well as enzyme recycling can potentially support the sustainable production of food-grade oligosaccharides from forest biomass.

Thermophilic enzyme systems for efficient conversion of lignocellulose to valuable products: Structural insights and future perspectives for esterases and oxidative catalysts.

Thermophilic enzyme systems are of major importance nowadays in all industrial processes due to their great performance at elevated temperatures. In the present review, an overview of the current knowledge on the properties of thermophilic and thermotolerant carbohydrate esterases and oxidative enzymes with great thermostability is provided, with respect to their potential use in biotechnological applications. A special focus is given to the lytic polysaccharide monooxygenases that are able to oxidatively cleave lignocellulose through the use of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. Structural characteristics of the enzymes, including active site conformation and surface properties are discussed and correlated with their substrate specificity and thermostability properties.

Effect of lignin fractions isolated from different biomass sources on cellulose oxidation by fungal lytic polysaccharide monooxygenases.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant lignocellulose in the presence of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. One of the possible systems that provide electrons to the LPMOs active site and promote the polysaccharide degradation involves the mediation of phenolic agents, such as lignin, low-molecular-weight lignin-derived compounds and other plant phenols. In the present work, the interaction of the bulk insoluble lignin fraction extracted from pretreated biomass with LPMOs and the ability to provide electrons to the active site of the enzymes is studied. RESULTS: The catalytic efficiency of three LPMOs, namely MtLPMO9 with C1/C4 regioselectivity, PcLPMO9D which is a C1 active LPMO and NcLPMO9C which is a C4 LPMO, was evaluated in the presence of different lignins. It was correlated with the physicochemical and structural properties of lignins, such as the molecular weight and the composition of aromatic and aliphatic hydroxyl groups. Moreover, the redox potential of lignins was determined with the use of large amplitude Fourier Transform alternating current cyclic voltammetry method and compared to the formal potential of the Cu (II) center in the active site of the LPMOs, providing more information about the lignin-LPMO interaction. The results demonstrated the existence of low-molecular weight lignin-derived compounds that are diffused in the reaction medium, which are able to reduce the enzyme active site and subsequently utilize additional electrons from the insoluble lignin fraction to promote the LPMO oxidative activity. Regarding the bulk lignin fractions, those isolated from the organosolv pretreated materials served as the best candidates in supplying electrons to the soluble compounds and, finally, to the enzymes. This difference, based on biomass pretreatment, was also demonstrated by the activity of LPMOs on natural substrates in the presence and absence of ascorbic acid as additional reducing agent. CONCLUSIONS: Lignins can support the action of LPMOs and serve indirectly as electron donors through low-molecular-weight soluble compounds. This ability depends on their physicochemical and structural properties and is related to the biomass source and pretreatment method.

Formation of Lignin Nanoparticles by Combining Organosolv Pretreatment of Birch Biomass and Homogenization Processes.

Valorization of lignocellulosic biomass into a biorefinery scheme requires the use of all biomass components; in this, the lignin fraction is often underutilized. Conversion of lignin to nanoparticles is an attractive solution. Here, we investigated the effect of different lignin isolation processes and a post-treatment homogenization step on particle formation. Lignin was isolated from birch chips by using two organosolv processes, traditional organosolv (OS) and hybrid organosolv-steam explosion (HOS-SE) at various ethanol contents. For post-treatment, lignin was homogenized at 500 bar using different ethanol:water ratios. Isolation of lignin with OS resulted in unshaped lignin particles, whereas after HOS-SE, lignin micro-particles were formed directly. Addition of an acidic catalyst during HOS-SE had a negative impact on the particle formation, and the optimal ethanol content was 50⁻60% v/v. Homogenization had a positive effect as it transformed initially unshaped lignin into spherical nanoparticles and reduced the size of the micro-particles isolated by HOS-SE. Ethanol content during homogenization affected the size of the particles, with the optimal results obtained at 75% v/v. We demonstrate that organosolv lignin can be used as an excellent starting material for nanoparticle preparation, with a simple method without the need for extensive chemical modification. It was also demonstrated that tuning of the operational parameters results in nanoparticles of smaller size and with better size homogeneity.

Fine-Tuned Enzymatic Hydrolysis of Organosolv Pretreated Forest Materials for the Efficient Production of Cellobiose.

Non-digestible oligosaccharides (NDOs) are likely prebiotic candidates that have been related to the prevention of intestinal infections and other disorders for both humans and animals. Lignocellulosic biomass is the largest carbon source in the biosphere, therefore cello-oligosacharides (COS), especially cellobiose, are potentially the most widely available choice of NDOs. Production of COS and cellobiose with enzymes offers numerous benefits over acid-catalyzed processes, as it is milder, environmentally friendly and produces fewer by-products. Cellobiohydrolases (CBHs) and a class of endoglucanases (EGs), namely processive EGs, are key enzymes for the production of COS, as they have higher preference toward glycosidic bonds near the end of cellulose chains and are able to release soluble products. In this work, we describe the heterologous expression and characterization of two CBHs from the filamentous fungus Thermothelomyces thermophila, as well as their synergism with proccessive EGs for cellobiose release from organosolv pretreated spruce and birch. The properties, inhibition kinetics and substrate specific activities for each enzyme are described in detail. The results show that a combination of EGs belonging to Glycosyl hydrolase families 5, 6, and 9, with a CBHI and CBHII in appropriate proportions, can enhance the production of COS from forest materials, underpinning the potential of these biocatalysts in the production of NDOs.

Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates.

BACKGROUND: Filamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates. RESULTS: In the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced MtEG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed. CONCLUSIONS: The endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported.

Effect of Different Pretreatment Methods on Birch Outer Bark: New Biorefinery Routes.

A comparative study among different pretreatment methods used for the fractionation of the birch outer bark components, including steam explosion, hydrothermal and organosolv treatments based on the use of ethanol/water media, is reported. The residual solid fractions have been characterized by ATR-FTIR, (13)C-solid-state NMR and morphological alterations after pretreatment were detected by scanning electron microscopy. The general chemical composition of the untreated and treated bark including determination of extractives, suberin, lignin and monosaccharides was also studied. Composition of the residual solid fraction and relative proportions of different components, as a function of the processing conditions, could be established. Organosolv treatment produces a suberin-rich solid fraction, while during hydrothermal and steam explosion treatment cleavage of polysaccharide bonds occurs. This work will provide a deeper fundamental knowledge of the bark chemical composition, thus increasing the utilization efficiency of birch outer bark and may create possibilities to up-scale the fractionation processes.