Abstract Background In this work, three pretreatments under investigation at the DOE Bioenergy Research Centers (BRCs) were subjected to a side-by-side comparison to assess their performance on model bioenergy hardwoods (a eucalyptus and a hybrid poplar). These include co-solvent-enhanced lignocellulosic fractionation (CELF), pretreatment with an ionic liquid using potentially biomass-derived components (cholinium lysinate or [Ch][Lys]), and two-stage Cu-catalyzed alkaline hydrogen peroxide pretreatment (Cu-AHP). For each of the feedstocks, the pretreatments were assessed for their impact on lignin and xylan solubilization and enzymatic hydrolysis yields as a function of enzyme loading. Lignins recovered from the pretreatments were characterized for polysaccharide content, molar mass distributions, β-aryl ether content, and response to depolymerization by thioacidolysis. Results All three pretreatments resulted in significant solubilization of lignin and xylan, with the CELF pretreatment solubilizing the majority of both biopolymer categories. Enzymatic hydrolysis yields were shown to exhibit a strong, positive correlation with the lignin solubilized for the low enzyme loadings. The pretreatment-derived solubles in the [Ch][Lys]-pretreated biomass were presumed to contribute to inhibition of enzymatic hydrolysis in the eucalyptus as a substantial fraction of the pretreatment liquor was carried forward into hydrolysis for this pretreatment. The pretreatment-solubilized lignins exhibited significant differences in polysaccharide content, molar mass distributions, aromatic monomer yield by thioacidolysis, and β-aryl ether content. Key trends include a substantially higher polysaccharide content in the lignins recovered from the [Ch][Lys] pretreatment and high β-aryl ether contents and aromatic monomer yields from the Cu-AHP pretreatment. For all lignins, the 13 C NMR-determined β-aryl ether content was shown to be correlated with the monomer yield with a second-order functionality. Conclusions Overall, it was demonstrated that the three pretreatments highlighted in this study demonstrated uniquely different functionalities in reducing biomass recalcitrance and achieving higher enzymatic hydrolysis yields for the hybrid poplar while yielding a lignin-rich stream that may be suitable for valorization. Furthermore, modification of lignin during pretreatment, particularly cleavage of β-aryl ether bonds, is shown to be detrimental to subsequent depolymerization.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Cytochrome c oxidase requires multiple heme and copper cofactors to catalyze the reduction of molecular oxygen to water. Although significant progress has been made in understanding the transport and incorporation of the copper ions, considerably less is known about the trafficking and insertion of the heme cofactors. Heme O synthase (HOS) and heme A synthase (HAS) from Rhodobacter sphaeroides (Cox10 and Cox15, respectively) and Bacillus subtilis (CtaB and CtaA, respectively) have been cloned and expressed in Escherichia coli. Our results demonstrate that HOS copurifies with HAS and that HAS copurifies with HOS, indicating that HOS and HAS interact and may form a physiologically relevant complex in vivo. Consistent with this hypothesis, the presence of HAS alters the total level of farnesylated hemes, providing further evidence that HOS and HAS interact. Our current working model is that HOS and HAS form a complex and that heme O is transferred directly from HOS to HAS. Because of the strong sequence similarity and evolutionary relationship between R. sphaeroides and mitochondria, our data suggest that this complex may form in eukaryotes as well.
A copper(II) macrocycle functions as an artificial ribonuclease. Cu([9]aneN3)Cl2 hydrolyzes both a 20-base RNA hairpin oligonucleotide and a 31-base sequence of TAR, an RNA sequence found in HIV. The cleavage reaction does not depend on interactions with specific nucleotide bases, and both single-stranded and double-stranded regions of the RNA are cleaved. Moreover, the cleavage of RNA by Cu([9]aneN3)Cl2 proceeds via a hydrolytic mechanism.
Abstract. Soil microbial processes, stimulated by agricultural fertilization, account for 90 % of anthropogenic nitrous oxide (N2O), the leading source of ozone depletion and a potent greenhouse gas. Efforts to reduce N2O flux commonly focus on reducing fertilization rates. Management of microbial processes responsible for N2O production may also be used to reduce N2O emissions, but this requires knowledge of the prevailing process. To this end, stable isotopes of N2O have been applied to differentiate N2O produced by nitrification and denitrification. To better understand the factors contributing to isotopic variation during denitrification, we characterized the δ15N, δ18O and site preference (SP; the intramolecular distribution of 15N in N2O) of N2O produced during NO3- reduction by Pseudomonas chlororaphis subsp. aureofaciens and P. c. subsp. chlororaphis. The terminal product of denitrification for these two species is N2O because they lack the gene nitrous oxide reductase, which is responsible for the reduction of N2O to N2. In addition to species, treatments included electron donor (citrate and succinate) and electron donor concentration (0.01, 0.1, 1 and 10 mM) as factors. In contrast to the expectation of a Rayleigh model, all treatments exhibited curvilinear behaviour between δ15N or δ18O and the extent of the reaction. The curvilinear behaviour indicates that the fractionation factor changed over the course of the reaction, something that is not unexpected for a multi-step process such as denitrification. Using the derivative of the equation, we estimated that the net isotope effects (η) vary by as much as 100 ‰ over the course of a single reaction, presenting challenges for using δ15N and δ18O as apportionment tools. In contrast, SP for denitrification was not affected by the extent of the reaction, the electron donor source or concentration, although the mean SP of N2O produced by each species differed. Therefore, SP remains a robust indicator of the origin of N2O. To improve apportionment estimates with SP, future studies could evaluate other factors that contribute to the variation in SP.
Heme A is an obligatory cofactor in all eukaryotic and many prokaryotic cytochrome c oxidase (CcO) enzymes. Despite its obvious importance to CcO and the electron transport pathway, essentially nothing is known concerning the regulation of heme A. Because CcO is the only natural target for heme A and copper is also required for CcO activity, it was postulated that copper might regulate heme A homeostasis. Work reported previously demonstrated that there is often a strong connection between copper and iron homeostasis in general, and circumstantial evidence pointed to a possible specific link between copper and heme A. To address this question, we conducted experiments to determine rigorously whether copper plays a role in heme A homeostasis. The two enzymes responsible for the conversion of heme B to heme A, heme O synthase (HOS) and heme A synthase (HAS), were separately genomically epitope-tagged in Saccharomyces cerevisiae, and their expression under various copper conditions was quantified by Western blot analysis. These results demonstrated that the sum of transcription, translation, and stability of HOS and HAS were independent of copper. Additionally, the effects of intracellular copper concentrations on the activity of HOS and HAS from Bacillus subtilis (expressed in Escherichia coli) and Rhodobacter sphaeroides were examined by analysis of cellular heme extracts. No trends with respect to intracellular copper were observed. In combination, our results demonstrate that intracellular copper levels do not affect the transcription, translation, stability, or activity of either HOS or HAS.
Divanillin (DV), which can be facilely synthesized via vanillin dimerization, was employed as a building block to formulate epoxy resin. DV was synthesized through a novel approach in hot water in only 30 min with a yield of 87.5%. The process involved FeSO4-catalyzed Na2S2O8-based oxidative coupling of vanillin without any purification, followed by treatment with biobased epichlorohydrin. Epoxidized-divanillin (EDV) was cured with the petroleum-based, commercially available hardener isophorone diamine (IPDA) and a biobased-diamine (GX-3090). Complete curing of the mixture was confirmed by Fourier transform infrared (FTIR) spectroscopy and statistical heat resistant-indices (Ts), which indicated the formation of cross-linked networks with a thermostability similar to materials prepared with diglycidyl ether bisphenol A (DGEBA, the commercial BPA-based resin). The epoxy resin developed with this new formulation had comparable storage moduli (1.7–2.3 GPa) and similar glass transition temperatures as commercial resins. The epoxy networks exhibited good solvent resistance, while the presence of aldehyde groups in EDV yielded in more readily cleavable ester and amide bonds during the cross-linking process, yielding a resin with improved degradation under acidic conditions. Almost 40% of the segments in networks cured with EDV/IPDA were solubilized in acetone after treatment with 1 M HCl at room temperature in 24 h.
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