Full Catalytic Activity Research Articles

Ni Single‐Atoms Supported on N‐Doped Carbon Prepared by Cation Exchange: Ultrafast Catalyst For The Reduction of Nitroarene Compounds Under Ambient Conditions

AbstractWe present a highly efficient single‐atom catalyst (SACs) tailored for nitroarene reduction using NaBH4 under ambient conditions. Employing an unique approach, we harnessed NaCl under high temperature to create host materials enriched with negatively charged nitrogenous and oxygenated functional groups, capable of anchoring and stabilizing Ni ions within the aromatic structure. The nickel single sites were prepared by a straightforward cation exchange method. STEM‐HAADF imaging confirmed the presence of nickel single‐atoms, while XPS, FTIR, and Raman spectra validated nickel coordination within the catalyst. Remarkably, the CN−Ni catalyst exhibited exceptional catalytic performance under ambient conditions, achieving a high catalytic activity with NaBH4 (TOF 2246 452 h−1 and 107 47 mmol g−1 min−1). It also demonstrated remarkable conversion exceeding 90 % and outstanding selectivity. Equally impressive was its ability to maintain full catalytic activity over multiple reaction cycles, highlighting its robustness. This work is a significant leap in SACs design, offering a versatile and highly effective preparation method for SACs based on N‐doped carbon with far‐reaching implications in industrial reduction reactions.

Structure–activity studies of Streptococcus pyogenes enzyme SpyCEP reveal high affinity for CXCL8 in the SpyCEP C-terminal

The Streptococcus pyogenes cell envelope protease (SpyCEP) is vital to streptococcal pathogenesis and disease progression. Despite its strong association with invasive disease, little is known about enzymatic function beyond the ELR+ CXC chemokine substrate range. As a serine protease, SpyCEP has a catalytic triad consisting of aspartate (D151), histidine (H279), and serine (S617) residues which are all thought to be mandatory for full activity. We utilised a range of SpyCEP constructs to investigate the protein domains and catalytic residues necessary for enzyme function. We designed a high-throughput mass spectrometry assay to measure CXCL8 cleavage and applied this for the first time to study the enzyme kinetics of SpyCEP. Results revealed a remarkably low Michaelis-Menton constant (KM) of 82 nM and a turnover of 1.65 molecules per second. We found that an N-terminally-truncated SpyCEP C-terminal construct containing just the catalytic dyad of H279 and S617 was capable of cleaving CXCL8 with a similar KM of 55 nM, albeit with a reduced substrate turnover of 2.7 molecules per hour, representing a 2200-fold reduction in activity. We conclude that the SpyCEP C-terminus plays a key role in high affinity substrate recognition and binding, but that the N-terminus is required for full catalytic activity.

Klebsiella phage KP34gp57 capsular depolymerase structure and function: from a serendipitous finding to the design of active mini-enzymes against K. pneumoniae.

In this work, we determined the structure of Klebsiella phage KP34p57 capsular depolymerase and dissected the role of individual domains in trimerization and functional activity. The crystal structure serendipitously revealed that the enzyme can exist in a monomeric state once deprived of its C-terminal domain. Based on the crystal structure and site-directed mutagenesis, we localized the key catalytic residues in an intra-subunit deep groove. Consistently, we show that C-terminally trimmed KP34p57 variants are monomeric, stable, and fully active. The elaboration of monomeric, fully active phage depolymerases is innovative in the field, as no previous example exists. Indeed, mini phage depolymerases can be combined in chimeric enzymes to extend their activity ranges, allowing their use against multiple serotypes.

Electrochemical recycling of homogeneous catalysts.

Homogeneous catalysts have rapid kinetics and keen reaction selectivity. However, their widespread use for industrial catalysis has remained limited because of challenges in reusability. Here, we propose a redox-mediated electrochemical approach for catalyst recycling using metallopolymer-functionalized electrodes for binding and release. The redox platform was investigated for the separation of key platinum and palladium homogeneous catalysts used in organic synthesis and industrial chemical manufacturing. Noble metal catalysts for hydrosilylation, silane etherification, Suzuki cross-coupling, and Wacker oxidation were recycled electrochemically. The redox electrodes demonstrated high sorption uptake for platinum-based catalysts (Qmax up to 200 milligrams of platinum per gram of adsorbent) from product mixtures, with up to 99.5% recovery, while retaining full catalytic activity over multiple cycles. The combination of mechanistic studies and electronic structure calculations indicate that selective interactions with anionic intermediates during the catalytic cycle played a key role in the separations. Last, continuous flow cell studies support the scalability and favorable technoeconomics of electrochemical recycling.

Dissecting the Kinetic Mechanism of Human Lysine Methyltransferase 2D and Its Interactions with the WRAD2 Complex.

Human lysine methyltransferase 2D (hKMT2D) is an epigenetic writer catalyzing the methylation of histone 3 lysine 4. hKMT2D by itself has little catalytic activity and reaches full activation as part of the WRAD2 complex, additionally comprising binding partners WDR5, RbBP5, Ash2L, and DPY30. Here, a detailed mechanistic study of the hKMT2D SET domain and its WRAD2 interactions is described. We characterized the WRAD2 subcomplexes containing full-length components and the hKMT2D SET domain. By performing steady-state analysis as a function of WRAD2 concentration, we identified the inner stoichiometry and determined the binding affinities for complex formation. Ash2L and RbBP5 were identified as the binding partners critical for the full catalytic activity of the SET domain. Contrary to a previous report, product and dead-end inhibitor studies identified hKMT2D as a rapid equilibrium random Bi–Bi mechanism with EAP and EBQ dead-end complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) analysis showed that hKMT2D uses a distributive mechanism and gives further insights into how the WRAD2 components affect mono-, di-, and trimethylation. We also conclude that the Win motif of hKMT2D is not essential in complex formation, unlike other hKMT2 proteins.

Rational Design of a Calcium-Independent Trypsin Variant

Trypsin is a long-known serine protease widely used in biochemical, analytical, biotechnological, or biocatalytic applications. The high biotechnological potential is based on its high catalytic activity, substrate specificity, and catalytic robustness in non-physiological reaction conditions. The latter is mainly due to its stable protein fold, to which six intramolecular disulfide bridges make a significant contribution. Although trypsin does not depend on cofactors, it essentially requires the binding of calcium ions to its calcium-binding site to obtain complete enzymatic activity and stability. This behavior is inevitably associated with a limitation of the enzyme’s applicability. To make trypsin intrinsically calcium-independent, we removed the native calcium-binding site and replaced it with another disulfide bridge. The resulting stabilized apo-trypsin (aTn) retains full catalytic activity as proven by enzyme kinetics. Studies using Ellmann’s reagent further prove that the two inserted cysteines at positions Glu70 and Glu80 are in their oxidized state, creating the desired functional disulfide bond. Furthermore, aTn is independent of calcium ions, possesses increased thermal and functional stability, and significantly reduced autolysis compared to wildtype trypsin. Finally, we confirmed our experimental data by solving the X-ray crystal structure of aTn.

Evolutionary origins of redox sensitivity in glucokinases

Glucokinase (GCK) plays a key role in regulating glucose homeostasis in the human body. Mammalian GCKs are particularly sensitive to the redox state of their environment and require high concentrations of reductant to maintain full catalytic activity. This observation has led to the proposal that redox regulation of mammalian GCKs may play an important role in controlling glucose metabolism in vivo. To investigate the origins of GCK’s redox sensitivity, we used ancestral sequence reconstruction to resurrect extinct proteins that existed along the vertebrate evolutionary trajectory.

Polyoxometalate-Mediated Vacancy-Engineered Cerium Oxide Nanoparticles Exhibiting Controlled Biological Enzyme-Mimicking Activities.

The biological enzyme-mimetic activity of cerium oxide nanoparticles (CeNPs) is well known to scavenge the reactive oxygen and nitrogen species in cell culture and animal models, imparting protection from the deleterious effects of oxidative and nitrosative stress. The superoxide dismutase (SOD)- and catalase-mimicking activity of CeNPs is reported to be controlled by the oxidation state of the surface "Ce" ions, where a high ratio of Ce3+/4+ or Ce4+/3+ has been considered for the displayed SOD and catalase-like activity, respectively. However, the redox behavior of CeNPs can be controlled by certain ligands that could offer changes in their enzyme-mimetic properties. Therefore, in this work, we have studied the enzyme-mimetic activities of CeNPs under the influence of polyoxometalates [phosphomolybdic acid (PMA) and phosphotungstic acid (PTA)], which are electron-dense molecules displaying quick and reversible multielectron redox reactions. Results revealed that the interaction of PMA with CeNPs results in the inhibition of the SOD-like activity; however, it has no impact on the catalase-like activity. Contrary to this, the interaction of PTA with CeNPs improved the SOD as well as catalase-like activities of CeNPs (3+), which generally do not exhibit catalase activity in the bare form. Although CeNPs (3+) did not show any peroxidase-like activity, CeNPs (4+) showed excellent activity, which was enhanced after the interaction with polyoxometalates. Further, the autoregeneration ability of CeNPs was found to be intact even after PTA or PMA interaction; however, the full catalytic activity was observed in the case of PTA but partially with PMA.

Comprehensive substrate specificity map of the mycobacterial serine hydrolase, LipN

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), uses a battery of lipases to scavenge host cell lipids and maintain an infection in its host. With their central roles in infection, lipases have become a viable target for drug development, but many of the lipases in M. tuberculosis remain poorly characterized. Our goal was to determine the substrate specificity, biochemical properties, and potential structural information of LipN, a proposed mycobacterial lipase. Initially, wild type LipN was heterologously expressed, purified to homogeneity, and its substrate specificity characterized against a library of 35 fluorogenic ester substrates. Wild type LipN demonstrated the highest catalytic efficiency against small carbon esters, including methyl ether, ethyl ether, and oxazole esters. LipN's preference for short (three carbons or less) esters with polar substituents suggests its physiological role as an esterase rather than a lipase and a selectivity for polar metabolites. Substitutional mutagenesis across the modeled active site and binding pocket of LipN allowed identification of the essential catalytic serine and histidine residues with the catalytic aspartate playing a less essential role in catalysis. The rest of the binding pocket showcased a range of polar residues required for full catalytic activity and two conserved glycines as the oxyanion hole. Herein, we showed that LipN likely catalyzes the ester hydrolysis of small, polar metabolites potentially aiding in the acquisition of additional energy sources from a host.

Chiral Amphiphilic Secondary Amine-Porphyrin Hybrids for Aqueous Organocatalysis.

Two chiral proline-derived amphiphilic 5-substituted-10,15,20-tris(4-sulfonatophenyl)porphyrins were prepared, and their pH-dependent supramolecular behavior was studied. In neutral aqueous solutions, the free-base form of the hybrids is highly soluble, allowing enamine-based organocatalysis to take place, whereas under acidic conditions, the porphyrinic protonated core of the hybrid leads to the formation of self-assembled structures, so that the hybrids flocculate and their catalytic activity is fully suppressed. The low degree of chirality transfer observed for aqueous Michael and aldol reactions strongly suggests that these reactions take place under true “in water” organocatalytic conditions. The highly insoluble catalyst aggregates can easily be separated from the reaction products by centrifugation of the acidic reaction mixtures, and after neutralization and desalting, the sodium salts of the sulfonated amine-porphyrin hybrids, retaining their full catalytic activity, can be recovered in high yield.

Ethanol conversion to selective high-value hydrocarbons over Ni/HZSM-5 zeolite catalyst

Bio-ethanol is a renewable fuel and can be converted to high-values chemicals. This study showed that more aromatics and gas products could be obtained at a low weight hourly space velocity (WHSV) than that at high WHSV using HZSM-5 and Ni/HZSM-5 catalysts. A low WHSV offered an increased tendency to build up aromatics and paraffins. Real bio-ethanol broth was tested and gave the similar result as pure ethanol solution. That catalysts could retain full catalytic activity after 168 h in reaction provides evidence for their long stability. This study advantageously demonstrates a promising approach to generate high-value products from bio-ethanol.

Multiple Sulfatase Deficiency: A Disease Comprising Mucopolysaccharidosis, Sphingolipidosis, and More Caused by a Defect in Posttranslational Modification.

Multiple sulfatase deficiency (MSD, MIM #272200) is an ultra-rare disease comprising pathophysiology and clinical features of mucopolysaccharidosis, sphingolipidosis and other sulfatase deficiencies. MSD is caused by impaired posttranslational activation of sulfatases through the formylglycine generating enzyme (FGE) encoded by the sulfatase modifying factor 1 (SUMF1) gene, which is mutated in MSD. FGE is a highly conserved, non-redundant ER protein that activates all cellular sulfatases by oxidizing a conserved cysteine in the active site of sulfatases that is necessary for full catalytic activity. SUMF1 mutations result in unstable, degradation-prone FGE that demonstrates reduced or absent catalytic activity, leading to decreased activity of all sulfatases. As the majority of sulfatases are localized to the lysosome, loss of sulfatase activity induces lysosomal storage of glycosaminoglycans and sulfatides and subsequent cellular pathology. MSD patients combine clinical features of all single sulfatase deficiencies in a systemic disease. Disease severity classifications distinguish cases based on age of onset and disease progression. A genotype- phenotype correlation has been proposed, biomarkers like excreted storage material and residual sulfatase activities do not correlate well with disease severity. The diagnosis of MSD is based on reduced sulfatase activities and detection of mutations in SUMF1. No therapy exists for MSD yet. This review summarizes the unique FGE/ sulfatase physiology, pathophysiology and clinical aspects in patients and their care and outlines future perspectives in MSD.

A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.

Comprehensive substrate specificity map of the mycobacterial serine hydrolase, LipN

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), uses a battery of lipases to scavenge host cell lipids and maintain an infection in its host. With their central roles in infection, lipases have become a viable target for drug development, but many of the lipases in M. tuberculosis remain poorly characterized. Our goal was to determine the substrate specificity, biochemical properties, and potential structural information of LipN, a proposed mycobacterial lipase. Initially, wild type LipN was heterologously expressed, purified to homogeneity, and its substrate specificity characterized against a library of 35 fluorogenic ester substrates. Wild type LipN demonstrated the highest catalytic efficiency against small carbon esters, including methyl ether, ethyl ether, and oxazole esters. LipN’s preference for short (three carbons or less) esters with polar substituents suggests its physiological role as an esterase rather than a lipase and a selectivity for polar metabolites. Substitutional mutagenesis across the modeled active site and binding pocket of LipN allowed identification of the essential catalytic serine and histidine residues with the catalytic aspartate playing a less essential role in catalysis. The rest of the binding pocket showcased a range of polar residues required for full catalytic activity and two conserved glycines as the oxyanion hole. Herein, we showed that LipN likely catalyzes the ester hydrolysis of small, polar metabolites potentially aiding in the acquisition of additional energy sources from a host.

Assembly of B4GALT1/ST6GAL1 heteromers in the Golgi membranes involves lateral interactions via highly charged surface domains

β-1,4-Galactosyltransferase 1 (B4GALT1) and ST6 β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) catalyze the successive addition of terminal β-1,4-linked galactose and α-2,6-linked sialic acid to N-glycans. Their exclusive interaction in the Golgi compartment is a prerequisite for their full catalytic activity, whereas a lack of this interaction is associated with cancers and hypoxia. To date, no structural information exists that shows how glycosyltransferases functionally assemble with each other. Using molecular docking simulations to predict interaction surfaces, along with mutagenesis screens and high-throughput FRET analyses in live cells to validate these predictions, we show here that B4GALT1 and ST6GAL1 interact via highly charged noncatalytic surfaces, leaving the active sites exposed and accessible for donor and acceptor substrate binding. Moreover, we found that the assembly of ST6GAL1 homomers in the endoplasmic reticulum before ST6GAL1 activation in the Golgi utilizes the same noncatalytic surface, whereas B4GALT1 uses its active-site surface for assembly, which silences its catalytic activity. Last, we show that the homomeric and heteromeric B4GALT1/ST6GAL1 complexes can assemble laterally in the Golgi membranes without forming cross-cisternal contacts between enzyme molecules residing in the opposite membranes of each Golgi cisterna. Our results provide detailed mechanistic insights into the regulation of glycosyltransferase interactions, the transitions between B4GALT1 and ST6GAL1 homo- and heteromers in the Golgi, and cooperative B4GALT1/ST6GAL1 function in N-glycan synthesis.

Triton X-100 or octyl glucoside inactivates acyl-CoA:cholesterol acyltransferase 1 by dissociating it from a two-fold dimer to a two-fold monomer

Cholesterol is an important lipid molecule and is needed for all mammalian cells. In various cell types, excess cholesterol is stored as cholesteryl esters; acyl-CoA:cholesterol acyltransferase 1 (ACAT1) plays an essential role in this storage process. ACAT1 is located at the endoplasmic reticulum and has nine transmembrane domains (TMDs). It is a member of the membrane-bound O-acyltransferase (MBOAT) family, in which members contain multiple TMDs and participate in a variety of biological functions. When solubilized in the zwitterionic detergent CHAPS, ACAT1 can be purified to homogeneity with full enzyme activity and behaves as a homotetrameric protein. ACAT1 contains two dimerization motifs. The first motif is located near the N-terminus and is not conserved in MBOATs. Deletion of the N-terminal dimerization domain converts ACAT1 to a dimer with full catalytic activity; therefore, ACAT1 is a two-fold dimer. The second dimerization domain, located near the C-terminus, is conserved in MBOATs; however, it was not known whether the C-terminal dimerization domain is required for enzyme activity. Here we show that treating ACAT1 with non-ionic detergent, Triton X-100 or octyl glucoside, causes the enzyme to become a two-fold monomer without any enzymatic activity. Detergent exchange of Triton X-100 with CHAPS restores ACAT1 to a two-fold dimer but fails to restore its enzymatic activity. These results implicate that ACAT1 requires hydrophobic subunit interactions near the C-terminus in order to remain active as a two-fold dimer. Our results also caution the use of Triton X-100 or octyl glucoside to purify other MBOATs.

Molecular Mechanism of Protein Kinase A in Fibrolamellar Hepatocellular Carcinoma

Fibrolamellar Hepatocellular Carcinoma (FL‐HCC) is a rare form of liver cancer that affects adolescents and young adults without a prior history of liver disease. Chemotherapy is ineffective in treating FL‐HCC, and surgical resection of the primary tumor and metastases is currently the only semi‐effective treatment. Reoccurrence rates in FL‐HCC are extremely high.A 400kB deletion in chromosome 19 results in the fusion of the first exon of a heat shock protein, DNAJB1, with the catalytic subunit of protein kinase A, PRKACA (1). The new chimeric protein, DNAJB1‐PRKACA, retains the full catalytic activity of protein kinase A. We have performed partial biophysical characterization of DNAJB1‐PRKACA in comparison to PRKACA. Our preliminary results indicate the structural stability of DNAJB1‐PRKACA may differ from that of PRKACA. We also report the DNAJB1‐PRKACA chimera forms a stable complex with its regulatory domains, and differences in structural stability may extend to the chimera/regulatory domain complex. Surface plasmon resonance (SPR) does not reveal any difference in the binding affinity of DNAJB1‐PRKACA and wild type PRKACA to their Regulatory subunit type Iβ.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Thiol-maleimide poly(ethylene glycol) crosslinking of L-asparaginase subunits at recombinant cysteine residues introduced by mutagenesis

L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin’s lymphoma. However, some disadvantages still limit its full application potential, e.g., allergic reactions, pancreatitis, and blood clotting impairment. Therefore, much effort has been directed at improving its performance. A popular strategy is to randomly conjugate L-asparaginase with mono-methoxy polyethylene glycol, which became a commercial FDA approved formulation widely used in recent years. To improve this formulation by PEGylation, herein we performed cysteine-directed conjugation of the L-asparaginase subunits to prevent dissociation-induced loss of activity. The recombinant cysteine conjugation sites were introduced by mutagenesis at surface-exposed positions on the protein to avoid affecting the catalytic activity. Three conjugates were obtained using different linear PEGs of 1000, 2000, and 5000 g/mol, with physical properties ranging from a semi-solid gel to a fully soluble state. The soluble-conjugate exhibited higher catalytic activity than the non-conjugated mutant, and the same activity than the native enzyme. The cysteine-directed crosslinking of the L-asparaginase subunits produced a higher molecular weight conjugate compared to the native tetrameric enzyme. This strategy might improve L-asparaginase efficiency for leukemia treatment by reducing glomerular filtration due to the increase in hydrodynamic size thus extending half-live, while at the same time retaining full catalytic activity.

Functional characterization of DYRK1A missense variants associated with a syndromic form of intellectual deficiency and autism.

ABSTRACTHaploinsufficiency of DYRK1A is a cause of a neurodevelopmental syndrome termed mental retardation autosomal dominant 7 (MRD7). Several truncation mutations, microdeletions and missense variants have been identified and result in a recognizable phenotypic profile, including microcephaly, intellectual disability, epileptic seizures, autism spectrum disorder and language delay. DYRK1A is an evolutionary conserved protein kinase which achieves full catalytic activity through tyrosine autophosphorylation. We used a heterologous mammalian expression system to explore the functional characteristics of pathogenic missense variants that affect the catalytic domain of DYRK1A. Four of the substitutions eliminated tyrosine autophosphorylation (L245R, F308V, S311F, S346P), indicating that these variants lacked kinase activity. Tyrosine phosphorylation of DYRK1A-L295F in mammalian cells was comparable to wild type, although the mutant showed lower catalytic activity and reduced thermodynamic stability in cellular thermal shift assays. In addition, we observed that one variant (DYRK1A-T588N) with a mutation outside the catalytic domain did not differ from wild-type DYRK1A in tyrosine autophosphorylation, catalytic activity or subcellular localization. These results suggest that the pathogenic missense variants in the catalytic domain of DYRK1A impair enzymatic function by affecting catalytic residues or by compromising the structural integrity of the kinase domain.This article has an associated First Person interview with the first author of the paper.

Hydrogen-enhanced catalytic hydrothermal gasification of biomass

The impact of hydrogen co-feeding on the catalytic hydrothermal gasification of wet biomass was explored in a continuous test rig using a feed of 10 wt% glycerol in water and a fixed bed of a carbon-supported ruthenium catalyst. The reactor was operated at a nominal temperature of 400 °C and at pressures of 26–28 MPa. Variation of the hydrogen-to-glycerol ratio as well as of the total pressure showed clearly the methanation reaction to be promoted at the expense of carbon dioxide and hydrogen formation. This is explained by a higher hydrogen surface coverage and consecutively higher rates for hydrogenation of surface-bound carbon. An increase in peak temperature of ca. 75 K occurred in the catalytic fixed-bed when co-feeding hydrogen. The measured product gas composition was close to the thermodynamic equilibrium calculated at the outlet temperature of the reactor. A maximum methane concentration of 86 vol% in the raw gas was obtained at 28 MPa with a stoichiometric addition of hydrogen. Full catalytic activity was maintained during and after the hydrogen co-feeding experiments, verified by comparing the performance of a run with a 10 wt% glycerol in water feed after co-feeding hydrogen, for which the product distribution was very close to the experiments before hydrogen co-feeding.