Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

表面活性剂对低渗透油藏渗吸的影响

采用不同类型的表面活性剂进行自发渗吸实验,并对表面活性剂改善岩石润湿性、降低界面张力的能力进行了分析。实验结果表明,阴离子型表面活性剂改善润湿性的能力好于其他类型的表面活性剂,且在岩心中的自发渗吸效果最好,这是由于阴离子型表面活性剂改善润湿性的机理为离子对形成机理,强于阳离子的吸附机理;接触角是决定渗吸能否发生的决定性因素,只有接触角小于70°时渗吸才能发生;界面张力影响渗吸速度和最终采出程度,对于渗透率为1 mD的岩心,最佳界面张力为10~(-1) mN/m。     

Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. Gd^III−Gd^III distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-Gd^III were indistinguishable from Gd^III−Gd^III distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.     

Biodegradability Study of the Biosurfactant Contained in a Crude Extract from Corn Steep Water

Over the last few years, the global biosurfactant market has raised due to the increasing awareness among consumers, for the use of biological or bio-based products. Because of their composition, it can be speculated that these are more biocompatible and more biodegradable than their chemical homologous. However, at the moment, no studies exist in the literature about the biodegradability of biosurfactants. In this work, a biosurfactant contained in a crude extract, obtained from a corn wet-milling industry stream that ferments spontaneously in the presence of lactic acid bacteria, was subjected to a biodegradation study, without addition of external microbial biomass, under different conditions of temperature (5–45 °C), biodegradation time (15–55 days), and pH (5–7). For that, a Box–Behnken factorial design was applied, which allowed to predict the percentage of biodegradation for the biosurfactant contained in the crude extract, between the range of the independent variables selected in the study, obtaining biodegradation values between 3 and 80%. The percentage of biodegradation for the biosurfactant was calculated based on the increase in the surface tension of samples of the crude extract. Furthermore, it was also possible to predict the variation in t_1/2 for the biosurfactant (time to achieve the 50% of biodegradation) under different conditions.     

Selective Analysis of Sulfur-Containing Species in a Heavy Crude Oil by Deuterium Labeling Reactions and Ultrahigh Resolution Mass Spectrometry

A heavy crude oil has been treated with deuterated alkylating reagents (CD₃I and C₂D₅I) and directly analyzed without any prior fractionation and chromatographic separation by high-field Orbitrap Fourier Transform Mass Spectrometry (FTMS) and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) using electrospray ionization (ESI). The reaction of a polycyclic aromatic sulfur heterocycles (PASHs) dibenzothiophene (DBT), in the presence of silver tetrafluoroborate (AgBF₄) with ethyl iodide (C₂H₅I) in anhydrous dichloroethane (DCE) was optimized as a sample reaction to study heavy crude oil mixtures, and the reaction yield was monitored and determined by proton nuclear magnetic resonance spectroscopy (¹H-NMR). The obtained conditions were then applied to a mixture of standard aromatic CH-, N-, O- and S-containing compounds and then a heavy crude oil, and only sulfur-containing compounds were selectively alkylated. The deuterium labeled alkylating reagents, iodomethane-d₃ (CD₃I) and iodoethane-d₅ (C₂D₅I), were employed to the alkylation of heavy crude oil to selectively differentiate the tagged sulfur species from the original crude oil.     

Beneficial effects of wettability altering surfactants in oil-wet fractured reservoirs

In fractured reservoirs, an effective matrix-fracture mass transfer is required for oil recovery. Surfactants have long been considered for oil recovery enhancement, mainly in terms of their ability to reduce oil–water interfacial tension. These surfactants are effective when the fractured formations are water-wet, where capillary imbibition of surfactants from the fracture into the matrix contributes to oil recovery. However, another beneficial aspect of surfactants, namely their ability to alter wettability, remains to be explored and exploited. Surfactants capable of altering wettability can be especially beneficial in oil-wet fractured formations, where the surfactant in the fracture diffuses into the matrix and alters the wettability, enabling imbibition of even more surfactant into the matrix. This sequential process of initial diffusion followed by imbibition continues well into the matrix yielding significant enhancements in oil recovery.In order to test this hypothesis of sequential diffusion–imbibition phenomenon, Dual-Drop Dual-Crystal (DDDC) contact angle experiments have been conducted using fractured Yates dolomite reservoir fluids, two types of surfactants (nonionic and anionic) and dolomite rock substrates. A new experimental procedure was developed in which crude oil equilibrated with reservoir brine has been exposed to surfactant to simulate the matrix-fracture interactions in fractured reservoirs. This procedure enables the measurements of dynamic contact angles and oil–water interfacial tensions, in addition to providing the visual observations of the dynamic behavior of crude oil trapped in the rock matrix as it encounters the diffusing surfactant from the fractures. Both the measurements and visual observations indicate wettability alterations of the matrix surface from oil-wet to less oil-wet or intermediate wet by the surfactants. Thus this study is of practical importance to oil-wet fractured formations where surfactant-induced wettability alterations can result in significant oil recovery enhancements. In addition, this study has also identified the need to include contact angle term in the dimensionless Bond number formulations for better quantitative interpretation of rock–fluids interactions.     

Synthesis and Interfacial Tension Behavior of Heavy Alkyl Benzene Sulfonates

Heavy alkyl benzene sulfonates (HABS) were synthesized, and their interfacial tensions were measured and compared with those of petroleum sulfonates ORS-41. Effects of their coupling with lignosulfonate were also studied. The results showed that HABS exhibited good interfacial activities and the system of HABS blending with LS displayed better interfacial activities than ORS-41 and therefore reduced the prime cost. Their best proportions of synergism were from 3:1 to 4:1. HABS might be a more promising alkali-surfactant-polymer (ASP) flooding agent.     

磺基琥珀酸双2-丁基辛酯钠盐的合成及表面活性测定

以马来酸酐和2-丁基辛醇为原料,经酯化得到相应的双酯,以乙醇作混相促进剂,使双酯与亚硫酸氢钠进行磺化反应,合成了磺基琥珀酸双2-丁基辛酯钠盐。采用正交试验对酯化反应条件进行了优化。合成产物的临界胶束浓度(cmc)值为3.58×10-6mol/L,表面张力(γcmc)为27.568mN/m。