Shale gas is becoming an increasingly important energy resource. In this study, the adsorption of methane on a dry, organic-rich Alum shale sample was studied at pressures up to ∼14 MPa and ...temperatures in the range 300–473 K, which are relevant to gas storage under geological conditions. Maximum methane excess uptake was 0.176–0.042 mmol g–1 (125–30 scf t–1) for the temperature range of 300–473 K. The decrease in maximum methane surface excess with increasing temperature can be described with a linear model. An isosteric enthalpy of adsorption 19.2 ± 0.1 kJ mol–1 was determined at 0.025 mmol g–1 using the van’t Hoff equation. Supercritical adsorption was modeled using the modified Dubinin–Radushkevich and the Langmuir equations. The results are compared with absolute isotherms calculated from surface excess and the pore volumes obtained from subcritical gas adsorption (nitrogen (78 K), carbon dioxide (273 and 195 K), and CH4 (112 K)). The subcritical adsorption and the surface excess results allow an upper limit to be put on the amount of gas that can be retained by adsorption during gas generation from petroleum source rocks.
Sorption capacities and pore characteristics of bulk shales and isolated kerogens have been determined for immature, oil-window, and gas-window mature samples from the Lower Toarcian Posidonia shale ...formation. Dubinin–Radushkevich (DR) micropore volumes, sorption pore volumes, and surface areas of shales and kerogens were determined from CO2 adsorption isotherms at −78 and 0 °C, and from N2 adsorption isotherms at −196 °C. Mercury injection capillary pressure porosimetry, grain density measurements, and helium pycnometry were used to determine shale and kerogen densities and total pore volumes. Total porosities decrease through the oil-window and then increase into the gas-window. High-pressure methane isotherms up to 14 MPa were determined at 45, 65, and 85 °C on dry shale and at 45 and 65 °C on kerogen. Methane excess uptakes at 65 °C and 11.5 MPa were in the range 0.056–0.110 mmol g–1 (40–78 scf t–1) for dry Posidonia shales and 0.36–0.70 mmol g–1 (253–499 scf t–1) for the corresponding dry kerogens. Absolute methane isotherms were calculated by correcting for the gas at bulk gas phase density in the sorption pore volume. The enthalpies of CH4 adsorption for shales and kerogens at zero surface coverage showed no significant variation with maturity, indicating that the sorption pore volume is the primary control on sorption uptake. The sum of pore volumes measured by (a) CO2 sorption at −78 °C and (b) mercury injection, are similar to the total porosity for shales. Since mercury in our experiments occupies pores with constrictions larger than ca. 6 nm, we infer that porosity measured by CO2 adsorption at −78 °C in the samples used in this study is largely within pores with effective diameters smaller than 6 nm. The linear correlation between maximum CH4 surface excess sorption and CO2 sorption pore volume at −78 °C is very strong for both shales and kerogens, and goes through the origin, suggesting that the vast majority of sorbed CH4 occurs in pores smaller than 6 nm. The DR micropore volume obtained from CO2 adsorption at 0 °C was 40%–62% of the corresponding CO2 sorption pore volume. Sorption mass balances using kerogen and shale isotherms showed that approximately half of the CO2 sorption in these dry shales is in organic matter, with the rest likely to be associated with the inorganic phase (mainly clay minerals). A similar distribution was observed for supercritical CH4 adsorption. Mass balances for adsorption isotherms for kerogen and clay minerals do not always account for the total measured sorbed CH4 on dry shales, suggesting that some sorption may not be completely accounted for by the minerals identified and kerogens in the shales.
Glycosylation of proteins is a key function of the biosynthetic‐secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking ...and signaling, cell‐cell adhesion, blood‐group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein‐based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose‐1‐phosphate‐guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1‐domain polyphosphate kinase 2 (1D‐Ppk2) expressed in E. coli for the cell‐free production and regeneration of GDP‐mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP‐mannose is produced at various conditions, that is pH 7–8, temperature 25–35°C and co‐factor concentrations of 5–20 mM MgCl2. The maximum reaction rate of GDP‐mannose achieved was 2.7 μM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP‐mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane‐deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER‐associated lipid‐linked oligosaccharide (LLO) assembly. Thereby, in a one‐pot reaction, phytanyl‐PP‐(GlcNAc)2‐Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl‐PP‐(GlcNAc)2‐Man1 can serve as a substrate for the synthesis of LLO for the cell‐free in vitro glycosylation of proteins. A high‐performance anion exchange chromatography method with UV and conductivity detection (HPAEC‐UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP‐mannose regenerating cascade and can further be used to study coupling of the GDP‐mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell‐free production of LLOs as precursors for in vitro glycoengineering of proteins.
A cell‐free cascade consisting of five enzymes expressed in E. coli for the synthesis and regeneration of GDP‐mannose, the main substrate in the buildup of lipid‐linked oligosaccharides in eukaryotes, was developed. The cascade was successfully coupled with a purified transmembrane‐deleted β‐1,4‐mannosyltransferase (ALG1ΔTM). A kinetic model was established to investigate inhibition in the network.
A cell-free cascade consisting of five enzymes expressed in E.coli for the synthesis of UDP-GlcNAc, an essential substrate for in vitro glycoengineering of proteins, was developed. UDP-GlcNAc was ...synthetized from low-cost substrates with a yield approaching 100%. The design of the cascade is complemented by steady state and kinetic analysis.
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•UDP-GlcNAc is produced from UMP, GlcNAc and polyphosphate with ATP regeneration in a cell-free cascade by five recombinant enzymes.•A yield approaching 100% is achieved at 40 °C and with a MgCl2 concentration of 45 mM.•The impact of temperature, co-factor (MgCl2) concentration and enzyme concentration on the UDP-GlcNAc flux has been elucidated.
In spite of huge endeavors in cell line engineering to produce glycoproteins with desired and uniform glycoforms, it is still not possible in vivo. Alternatively, in vitro glycoengineering can be used for the modification of glycans. However, in vitro glycoengineering relies on expensive nucleotide sugars, such as uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) which serves as GlcNAc donor for the synthesis of various glycans. In this work, we present a systematic study for the cell-free de novo synthesis and regeneration of UDP-GlcNAc from polyphosphate, UMP and GlcNAc by a cascade of five enzymes (N–acetylhexosamine kinase (NahK), Glc–1P uridyltransferase (GalU), uridine monophosphate kinase (URA6), polyphosphate kinase (PPK3), and inorganic diphosphatase (PmPpA). All enzymes were expressed in E. coli BL21 Gold (DE3) and purified using immobilized metal affinity chromatography (IMAC). Results from one-pot experiments demonstrate the successful production of UDP-GlcNAc with a yield approaching 100%. The highest volumetric productivity of the cascade was about 0.81 g L−1 h−1 of UDP-GlcNAc. A simple model based on mass action kinetics was sufficient to capture the dynamic behavior of the multienzyme pathway. Moreover, a design equation based on metabolic control analysis was established to investigate the effect of enzyme concentration on the UDP-GlcNAc flux and to demonstrate that the flux of UDP-GlcNAc can be controlled by means of the enzyme concentrations. The effect of temperature on the UDP-GlcNAc flux followed an Arrhenius equation and the optimal co-factor concentration (Mg2+) for high UDP-GlcNAc synthesis rates depended on the working temperature. In conclusion, the study covers the entire engineering process of a multienzyme cascade, i.e. pathway design, enzyme expression, enzyme purification, reaction kinetics and investigation of the influence of basic parameters (temperature, co-factor concentration, enzyme concentration) on the synthesis rate. Thus, the study lays the foundation for future cascade optimization, preparative scale UDP-GlcNAc synthesis and for in situ coupling of the network with UDP-GlcNAc transferases to efficiently regenerate UDP-GlcNAc. Hence, this study provides a further step towards cost-effective in vitro glycoengineering of antibodies and other glycosylated proteins.
Recombinant Leloir glycosyltransferases can be exploited to synthesize a wide range of HMOs using in vitro biocatalytic reactions. However, high costs and unavailability of bulk amounts of most ...nucleotide sugars, such as guanosine diphosphate L‐fucose (GDP‐Fuc), are major obstacles for the efficient large‐scale synthesis. Here, we report two novel multi‐enzyme cascades for the synthesis of GDP‐Fuc from readily available and low cost precursors. The first cascade was developed to produce GDP‐Fuc from guanosine (Guo), fucose (Fuc), polyphosphate (PolyPn) and catalytic amounts of adenine triphosphate (ATP). GDP‐Fuc was produced with a final concentration of 7 mM (4.1 g/L) and a reaction yield of 68 % from Guo and Fuc within 48 h with a biocatalyst load of 0.34 genzyme/gproduct. A second cascade, consisting of ten enzymes and eleven reactions was developed to carry out the synthesis from mannose (Man), Guo, PolyPn, L‐glutamine (L‐Glu) and catalytic amounts of ATP, and nicotinamide adenine dinucleotide phosphate (NADPH). Utilizing this cascade, GDP‐Fuc was produced with a final concentration of 7.6 mM (4.5 g/L) and a reaction yield of 72 % in a reaction time of 48 h with a biocatalyst load of 0.97 genzyme/gproduct. Finally, a method for chromatographic purification of GDP‐Fuc was established achieving product purities of 90.5 %.
Multi‐enzyme cascade reactions: In vitro enzyme cascades for the one‐pot synthesis of guanosine diphosphate fucose (GDP‐Fuc) from low cost and readily available substrates. In situ co‐factor regeneration was implemented and a robust and scalable ion exchange chromatography protocol for the purification of GDP‐Fuc from the reaction was established. The developed cascades and the purification protocol can be employed for the efficient biocatalytic production of GDP‐Fuc.
An inter-laboratory study of high-pressure gas sorption measurements on two carbonaceous shales has been conducted in order to assess the reproducibility of the sorption isotherms and identify ...possible sources of error. The measurements were carried out by seven international research laboratories using either in-house or commercial sorption equipment (manometric and gravimetric methods). Excess sorption isotherms for methane, carbon dioxide and ethane were measured at 65°C and at pressures up to 25MPa on two organic-rich shales in the dry state. The samples used in this study were taken from immature Posidonia shale (Germany) and over-mature Upper Chokier Formation (Belgium). Their total organic carbon (TOC) contents were 15.1% and 4.4% , respectively, and their vitrinite reflectance (VRr) values 0.5% and 2.0%.
The objective of this study was to assess the reproducibility of sorption isotherms among laboratories each following their own measurement and data reduction procedures. All labs were asked to follow a predefined sample drying procedure prior to measurement in order to minimize any effects related to moisture. The reproducibility of the methane excess sorption isotherms was better for the high-maturity shale (within 0.02–0.03mmol/g) than for the low-maturity sample (up to 0.1mmol/g), similar to observations in earlier inter-laboratory studies on coals. The reproducibility for CO2 and C2H6 sorption isotherms was satisfactory at pressures below 5MPa, however, the results deviate considerably at higher pressures. Anomalies in the shape of the excess sorption isotherms were observed for CO2 and C2H6 and these are explained as being due to high sensitivity of gas density to temperature and pressure close to the critical point as well as from a limited measurement accuracy and possibly uncertainty in the equation of state (EoS).
The low sorption capacity of carbonaceous shales (as compared to coals and activated carbons) sets very high demands on the accuracy of pressure and temperature measurement and precise temperature control. Furthermore, the sample treatment, measurement and data reduction procedures must be optimized in order to achieve satisfactory inter-laboratory consistency and accuracy. Systematic errors must be minimized first by calibrating the pressure and temperature sensors to high-quality standards. Blank sorption measurements with a non-sorbing sample (e.g. stainless steel) can be used to identify and quantitatively account for measuring artifacts resulting from unknown residual systematic errors or from the limited accuracy of the EoS. The possible sources of error causing the observed discrepancies are discussed.
•First inter-laboratory study of reproducibility of sorption isotherms on shales•Excess sorption isotherms of CH4, CO2 and C2H6 for low- and high-maturity shales•Significant discrepancies observed at high pressures (>5MPa)•Low sorption capacities of shales require the highest measurement standards.•Recommendations for improving accuracy in sorption measurement
High costs and low availability of UDP‐galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for ...the scalable, biocatalytic synthesis and purification of UDP‐galactose. UDP‐galactose was produced with a titer of 48 mM (27.2 g/L) in a small‐scale batch process (200 μL) within 24 h using 0.02 genzyme/gproduct. Through in‐situ ATP regeneration, the amount of ATP (0.6 mM) supplemented was around 240‐fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP‐galactose with a purity of 92 %. The synthesis was transferred to 1 L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4 g/L UDP‐galactose were produced within 23 h with a synthesis yield of 71 % and a biocatalyst load of 0.05 gtotal_protein/gproduct. The costs for substrates per gram of UDP‐galactose synthesized were around 0.26 €/g.
An enzymatic platform was developed for the synthesis of UDP‐Gal from low‐cost and readily available precursors. The overall process was optimized for efficiency by bypassing time‐consuming and expensive enzyme purification steps followed by a scale‐up into a 1 L bioreactor. Here, within 23 h, a total of 23.4 g/L of UDP‐galactose were produced. Costs for substrates per gram of UDP‐Gal synthesized were 0.26 €/g.
•Cell-free synthesis of lipid-linked mannopentaose-di-(N-acetylglucosamine) from a lipid-linked precursor, polyphosphate, mannose and GDP.•In-vitro N-glycosylation of peptides using purified ...oligosaccharyltransferase T. brucei STT3A.•GDP-mannose synthesis and regeneration cascade coupled to three mannosyltransferases.•Liquid chromatography - mass spectrometry (LC–MS) analysis of in-vitro N-glycosylated peptides.
A wide range of glycoproteins can be recombinantly expressed in aglycosylated forms in bacterial and cell-free production systems. To investigate the effect of glycosylation of these proteins on receptor binding, stability, efficacy as drugs, pharmacodynamics and pharmacokinetics, an efficient glycosylation platform is required. Here, we present a cell-free synthetic platform for the in vitro N-glycosylation of peptides mimicking the endoplasmic reticulum (ER) glycosylation machinery of eukaryotes. The one-pot, two compartment multi-enzyme cascade consisting of eight recombinant enzymes including the three Leloir glycosyltransferases, Alg1, Alg2 and Alg11, expressed in E. coli and S. cerevisiae, respectively, has been engineered to produce the core lipid-linked (LL) oligosaccharide mannopentaose-di-(N-acetylglucosamine) (LL-Man5). Pythanol (C20H42O), a readily available alcohol consisting of regular isoprenoid units, was utilized as the lipid anchor. As part of the cascade, GDP-mannose was de novo produced from the inexpensive substrates ADP, polyphosphate and mannose. To prevent enzyme inhibition, the nucleotide sugar cascade and the glycosyltransferase were segregated into two compartments by a cellulose ester membrane with 3.5 kDa cut-off allowing for the effective diffusion of GDP-mannose across compartments. Finally, as a proof-of-principle, pythanyl-linked Man5 and the single-subunit oligosaccharyltransferase Trypanosoma brucei STT3A expressed in Sf9 insect cells were used to in vitro N-glycosylate a synthetic peptide of ten amino acids bearing the eukaryotic consensus motif N-X-S/T.
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BioRender.com
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Cell-free, chemoenzymatic platforms are emerging technologies towards generating glycoconjugates with defined and homogeneous glycoforms. Recombinant ...oligosaccharyltransferases can be applied to glycosylate “empty,” i.e., aglycosyalted, peptides and proteins. While bacterial oligosaccharlytransferases have been extensively investigated, only recently a recombinant eukaryotic single-subunit oligosaccharyltransferase has been successfully used to
in vitro N
-glycosylate peptides. However, its applicability towards synthesizing full-length glycoproteins and utilizing glycans beyond mannose-type glycans for the transfer have not be determined. Here, we show for the first time the synthesis of hybrid- and complex-type glycans using synthetic lipid carriers as substrates for
in vitro N
-glycosylation reactions. For this purpose, transmembrane-deleted human β-1,2
N
-acetylglucosamintransferase I and II (MGAT1ΔTM and MGAT2ΔTM) and β-1,4-galactosyltransferase (GalTΔTM) have been expressed in
Escherichia coli
and used to extend an existing multi-enzyme cascade. Both hybrid and agalactosylated complex structures were transferred to the
N
-glycosylation consensus sequence of peptides (10 amino acids: G-S-D-A-N-Y-T-Y-T-Q) by the recombinant oligosaccharyltransferase STT3A from
Trypanosoma brucei
.
Shale gas is an important hydrocarbon resource in a global context. It has had a significant impact on energy resources in the US, but the worldwide development of this methane resource requires ...further research to increase the understanding of the relationship of shale structural characteristics to methane storage capacity. In this study a range of gas adsorption, microscopic, mercury injection capillary pressure porosimetry and pycnometry techniques were used to characterize the full range of porosity in a series of shales of different thermal maturity. Supercritical methane adsorption methods for shale under conditions which simulate geological conditions (up to 473 K and 15 MPa) were developed. These methods were used to measure the methane adsorption isotherms of Posidonia shales where the kerogen maturity ranged from immature, through oil window, to gas window. Subcritical methane and carbon dioxide adsorption studies were used for determining pore structure characteristics of the shales. Mercury injection capillary pressure porosimetry was used to characterize the meso and macro porosity of shales. The sum of the CO
2
sorption pore volume at 195 K and mercury injection capillary pressure pore volumes (1093–5.6 nm) were equal to the corresponding total pore volume (< 1093 nm) thereby giving an equation accounting for virtually all the available shale porosity. These measurements allowed quantification of all the available porosity in shales and were used for estimating the contributions of methane stored as ‘free’ compressed gas and as adsorbed gas to overall methane storage capacity of shales. Both the mineral and kerogen components of shale were studied by comparing shale and the corresponding isolated kerogens so that the relative contributions of these components could be assessed. The results show that the methane adsorption characteristics were much higher for the kerogens and represented 35–60% of the total adsorption capacity for the shales used in this study, which had total organic contents in range 5.8–10.9 wt%. Microscopy studies revealed that the pore systems in clay-rich, organic-rich and microfossil-rich parts of shale are very different, and also the importance of the inter-granular organic-mineral interface.
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