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Open Access
Abstract: Biofuels are critical drop-in replacement energy sources to support the decarbonisation of hard-to-abate sectors such as aviation and marine shipping. Transesterification of non-edible oils is a well-established route to biodiesel as a versatile liquid transport fuel, but is challenging to scale using existing homogeneous liquid base catalysts. In this work, we report the synthesis, characterisation, and application of silica-supported MgO solid base catalysts for triglyceride transesterification with methanol and highlight the impact of silica pore structure on performance. True liquid crystal templating enables the one-pot synthesis of mesoporous MgO/SBA-15 catalysts with variable Mg content, or hierarchical macroporous–mesoporous MgO/SBA-15 analogues through the addition of polystyrene nanospheres. Both MgO/SBA-15 families exhibit highly ordered pore networks; however, ~280 nm macropores stabilise Mg-O-Si interfacial species even at high Mg loading, in contrast to the mesoporous support that permits sintering of ~14 nm MgO nanocrystals. Hierarchical porous MgO/SBA-15 catalysts exhibit higher specific activity and conversion of tributyrin to methyl butyrate than their mesoporous analogues (3 mmol⋅h−1⋅g−1 versus 2 mmol⋅h−1⋅g−1 at 60 °C and 11 wt% Mg). The magnitude of this rate enhancement increases with triglyceride chain length, being approximately three-fold for trilaurin (C12) transesterification at 90 °C, attributed to superior in-pore mass transport of bulky reactants through the hierarchical porous catalyst.
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Nov 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32728]
Open Access
Abstract: Certain members of the bacterial cytochrome P450 152 family (CYP152) are peroxygenases that catalyse the decarboxylation of fatty acids into terminal olefins making them attractive biocatalysts for biofuel production. To date, the characterisation of decarboxylating CYP152s has mainly focused on their reaction with saturated fatty acid substrates. CYP152s are often co-purified with a bound substrate, which is generally removed before further experiments are conducted. In the present work we identified that heterologous over-expressed CYP152 from Staphylococcus aureus (OleTSa) is co-purified with the trans-monounsaturated C18:1 fatty acid, elaidic acid. We report the spectral, thermodynamic and kinetic characteristics of OleTSa bound to both elaidic acid and its saturated counterpart, stearic acid. Despite differing spectral profiles, metabolic and kinetic studies reveal that OleTSa is capable of decarboxylating elaidic acid, converting it to heptadeca-1,8-diene following addition of hydrogen peroxide, at the same rate and chemoselectivity as the conversion of stearic acid to 1-heptadecane. The X-ray crystal structure of the as purified OleTSa in complex with elaidic acid is also presented, allowing for several key residues to be identified for site-directed mutagenesis studies. The influence of the site-directed variants on C18:0 and C18:1 product formation, binding thermodynamics and kinetics have been investigated, showing that while spectral differences occur as a likely result of perturbing the binding pocket, this does not alter the chemoselectivity of the enzyme. Our work provides important insights into the mechanism of decarboxylation of an unsaturated fatty acid substrate by OleTSa potentially expanding the sustainable substrate space available for CYP152s.
Graphical abstract
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Oct 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31378]
Open Access
Abstract: Background: Cyanobacteria are promising platforms for metabolic engineering to convert carbon dioxide into valuable fuels and chemicals, addressing both energy demands and global climate change. Among various fuels and chemicals, isobutanol (IB) and 3-methyl-1-butanol (3M1B) have gained increasing attention due to their superior physical properties, such as high energy density, low water solubility, and low hygroscopicity. Heterologously expressing α-ketoisovalerate decarboxylase (KivdS286T) in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) enables microbial production of IB and 3M1B through the 2-keto acid pathway, with KivdS286T identified as a key bottleneck limiting production efficiency. Results: To address this limitation, a high-throughput screening method based on the consumption of the substrate 2-ketoisovalerate was successfully established. This screen was coupled with random mutagenesis, via error-prone PCR, of KivdS286T. Out of the 1600 variants, 1B12, featuring dual substitutions K419E and T186S, exhibited a 55% increase in IB production and a 50% increase in 3M1B production in Synechocystis on the fourth day of cultivation. The crystal structure of KivdS286T was determined as a tetramer with a resolution of 2.8 Å to provide a framework for analyzing the structural basis for the enhanced butanol production conferred by the K419E and T186S substitutions. Conclusions: A novel Kivd variant, 1B12, was successfully generated via directed evolution, with enhanced catalytic activity for microbial IB and 3M1B biosynthesis. To our knowledge, this study represents the first successful application of directed evolution on the rate-limiting enzyme of a specific metabolic pathway to enhance biochemical production in cyanobacteria.
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Jul 2025
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B18-Core EXAFS
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Diamond Proposal Number(s):
[29271]
Open Access
Abstract: Bioethanol upgrading to n-butanol is catalysed by homogeneous catalysts under mild solventless liquid phase conditions, at which heterogeneous catalysts have poor performance. Here a heterogeneous catalyst, initially comprised of a Ru(bipy) complex incorporated into a polyphenylene support, gave a catalytic performance (TON of 3565 Ru-1 and 82% n-butanol selectivity) that exceeded contemporary homogeneous and heterogeneous catalysts. Catalyst deactivation by water byproduct inhibition was improved by removal of water, via recharging the reactor with fresh ethanol, or by the co-addition of molecular sieve. The Ru polyphenylene catalyst showed greater stability in the presence of molecular sieve compared to the homogeneous catalyst, enabling high butanol yields. Characterisation showed a structural transformation of the single atom Ru(bipy) into 2.6 nm Ru(0) nanoparticles after 16 h reaction. While the Ru(0) had moderate recyclability (TON of 1000 Ru-1) the initial highly active species seen in the fresh catalyst are not stable.
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Jan 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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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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Dec 2024
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[20568]
Open Access
Abstract: Nanostructured materials can be utilised as potential catalysts for the production of platform chemicals and renewable biofuels from biomass derived molecules. The formation of hierarchical meso-microporous zeolites LTL and FAU via the surfactant assisted tandem acid-base post-synthesis treatment has been investigated by time-resolved in situ synchrotron SAXS and WAXS, providing a new insight into the mechanism of the mesostructuring treatment. Based on the results of TEM and in situ synchrotron measurements, a model for the formation of the core-shell structure of LTL zeolite crystals is proposed. Complementary evaluation using FTIR, NMR and nitrogen adsorption, in conjunction with reaction studies on mesostructured zeolites, demonstrated a potential for enhanced catalytic performance of these materials owing to the increased accessibility of the active sites and reduced transport limitations.
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Oct 2024
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[15705]
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.
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Jan 2024
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I20-Scanning-X-ray spectroscopy (XAS/XES)
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Diamond Proposal Number(s):
[28477]
Abstract: Dry plant matter (biomass) is an abundantly available raw material for the production of biofuels. The principal carbohydrate polymer it contains, cellulose, is packed with glucose units that can be fermented into bioethanol - a sustainable liquid fuel. These polymers are difficult to break down chemically, but we get a helping hand from the natural enzymes that have evolved to do the job. Widely found enzymes, lytic polysaccharide monooxygenases (LPMOs), are major contributors to natural carbon recycling and are now used in commercial bioethanol production. However, questions remain around how these enzymes survive the powerful chemistry they wield. In work recently published in the Journal of the American Chemical Society, researchers from the University of Manchester, Novozymes, Graz University of Technology, the University of York and Diamond Light Source, used a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, electron paramagnetic resonance spectroscopy and High Energy Resolution Fluorescence Detection X-ray Absorption Spectroscopy (HERFD−XAS) to investigate how these oxidative enzymes protect themselves from harmful side reactions. Their results show that short-lived molecules produced during the breakdown of polysaccharides provide a built-in defence and repair mechanism.
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Dec 2023
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Open Access
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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Oct 2023
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I20-Scanning-X-ray spectroscopy (XAS/XES)
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Jingming
Zhao
,
Ying
Zhuo
,
Daniel E.
Diaz
,
Muralidharan
Shanmugam
,
Abbey J.
Telfer
,
Peter J.
Lindley
,
Daniel
Kracher
,
Takahiro
Hayashi
,
Lisa S.
Seibt
,
Florence J.
Hardy
,
Oliver
Manners
,
Tobias M.
Hedison
,
Katherine A.
Hollywood
,
Reynard
Spiess
,
Kathleen M.
Cain
,
Sofia
Diaz-Moreno
,
Nigel S.
Scrutton
,
Morten
Tovborg
,
Paul H.
Walton
,
Derren J.
Heyes
,
Anthony P.
Green
Diamond Proposal Number(s):
[28477]
Open Access
Abstract: Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C–H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII–(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII–tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues “on the fly” to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.
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Sep 2023
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