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Abstract: Multifunctionality, processivity, and thermostability are critical for the cost-effective enzymatic saccharification of non-food plant biomass polymers such as β-glucans, celluloses, and xylans to generate biofuels and other valuable products. We present molecular insights into a processive multifunctional endo-1,3-1,4-β-D-glucanase (Tt_End5A) from the hyperthermophilic bacterium Thermogutta terrifontis. Tt_End5A demonstrated activities against a broad spectrum of β-polysaccharides, including barley glucan, lichenan, carboxymethyl cellulose, regenerated amorphous cellulose (RAC), Avicel, xylan, laminarin, mannan, curdlan, xanthan, and various chromogenic substrates at pH 7 and temperatures ranging from 70-80°C. The enzyme exhibited a high level of processivity on RAC and retained over 90% activity at 80 °C for an extended period, indicating exceptional thermal stability. The 1.20 Å crystal structure of the Tt_End5A catalytic domain revealed an archetypal glycoside hydrolase family 5 (GH5) catalytic TIM-(β/α)8-barrel, supplemented with additional β-strands, elongated α-helices, and a rare cis-non-Pro (His481-cis-Ala482) peptide. A large central cleft was observed in the 3D structure, which is likely related to the enzyme's multifunctionality and processivity. The catalytic domain is preceded by a novel N-terminal multivalent carbohydrate-binding module (CBM) that enhances the enzymatic degradation of insoluble polysaccharides. Mutagenesis studies, ligand interaction analyses, and the structurally conserved positions of E329 and E448 in Tt_End5A suggest that these residues function as the proton donor and nucleophile in the catalytic mechanism. Owing to its multifunctionality and processivity, Tt_End5A can reduce the need for multiple saccharification enzymes to generate fermentable sugars from plant biomass for bioethanol production. Additionally, it holds promise for applications in the pharmaceutical, feed, and food industries.

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Abstract: Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs.

Open Access

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Abstract: Synthesis of 2,5-furandicarboxylic acid (FDCA) can be achieved via catalytic oxidation of 5-hydroxymethylfurfural (5-HMF), in which both base and catalyst play important roles. This work presents the development of a simple synthesis method (based on a commercial parent 10 wt.% Pd/C catalyst) to prepare the bimetallic AuPd alloy catalysts (i. e., AuPd/C) for selective 5-HMF oxidation to FDCA. When using the strong base of NaOH, Pd and Au cooperate to promote FDCA formation when deployed either separately (as a physical mixture of the monometallic Au/C and Pd/C catalysts) or ideally alloyed (AuPd/C), with complete 5-HMF conversion and FDCA yields of 66 % vs 77 %, respectively. However, NaOH also promoted the formation of undesired by-products, leading to poor mass balances (<81 %). Comparatively, under weak base conditions (using NaHCO3), an increase in Au loading in the AuPd/C catalysts enhances 5-HMF conversion and FDCA productivity (due to the enhanced carbonyl oxidation capacity) which coincides with a superior mass balances of >97 %. Yet, the excessive Pd content in the AuPd/C catalysts was not beneficial in promoting FDCA formation.

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Abstract: Single atom electrocatalysts (SAECs) exhibit intriguing catalytic performance due to their utmost atom-utilization efficiency and unique electronic structure. However, the application of SAECs is limited on simple reactions such as the hydrogen/oxygen evolution reactions, and oxygen/nitrogen reduction reactions. Herein, single copper atoms supported on nitrogen doped carbon nanosheets (Cu/NCNSs) were demonstrated as a versatile SAEC for biomass upgrade. The activity of Cu/NCNSs was benchmarked against commercial Cu nanoparticles. Twelve basic platform compounds including 5-hydroxymethylfurfural (HMF), furfural, glucose, formaldehyde, methanol, ethanol, isopropanol, ethylene glycol, glycerol, benzyl alcohol, formic acid, and oxalic acid were employed as substrates. The products of electro-oxidation of these substrates were analyzed by high performance liquid chromatography. The reaction pathways and mechanisms of substrate oxidation on Cu/NCNSs and Cu NPs were explored by in-situ electrochemical Raman spectra. HMF oxidation on Cu NPs followed the 5-diformylfuran (DFF) path at lower potentials, while it turned to 5-hydroxymethyl-2-furancarboxylic acid path at applied potentials over 1.67 V. For Cu/NCNSs, HMF oxidation followed the alcohol hydroxyl oxidation pathway, that is, the direction of generating DFF. The active sites for substrate oxidation on Cu/NCNSs are different from those on Cu NPs. This work broadened the application of SAECs in the field of biomass upgrade, and paved a new route to fabricate advanced electrocatalysts for biomass valorization.

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Abstract: Many conversion technologies and processes for bioenergy generation from wastes have been reported and discussed in the previous chapters. These conversion technologies are being selected and applied depending on the type of wastes, chemical composition of the available waste and the desired energy vector. For example, anaerobic digestion (AD) is used for biogas production from mixed biological wastes with varied chemical compositions. Fermentation for bioethanol production is used for wastes that are rich in simple sugars and/or starch. Pyrolysis, combustion and gasification are used for crude bio-oil and/or syngas production from a wide range of wastes, especially those that contain high lignocellulosic compounds. These are environmentally friendly technologies for waste management and bioenergy production, but their economic feasibility is usually limited using a process of a single conversion route. Recent research and prospects suggested that integrating processes for bioenergy production from waste could increase the efficiency of the system in terms of economy, energy recovery and beneficial impact on the environment. This chapter discusses waste biorefinery as a recent trend towards circular bioeconomy. The chapter provides suggestions for future integrated systems for the simultaneous production of multiple energy vectors and high-value chemicals from different types of wastes. In the integrated system, the by-products from the first conversion process are used as substrates for the subsequent conversion process and so on. The importance of catalysis in offering flexibility in an integrated biorefinery system by providing novel routes and downstream environmental solutions for flue gas and exhaust gas cleaning was also covered in the chapter. The chapter concludes with a discussion of the role of models with bioenergy and biomass resource decision making, with focus on bioenergy from waste case studies. This includes assessment of the key issues that determine the economic feasibility and environmental impacts of feedstock choices and technology options. Also covering the social and political frameworks that will enable and drive transitions towards increased bioenergy from waste activities. The chapter presents policy case studies from the EU, China, USA and India, and highlights how social acceptance will be key to the success of any bioenergy from waste sector.