Enhanced expression and purification of fungal galactose oxidase in Escherichia coli and use for analysis of a saturation mutagenesis library

Galactose oxidase (GO) displays broad primary alcohol substrate specificity and so offers potential for engineering new substrate specificity by directed evolution. Producing variant libraries of sufficient complexity ideally requires expression of functional protein in a host such as Escherichia coli. Wild-type GO is produced by the fungus Fusarium graminiarum and is expressed poorly in E. coli. We introduced silent mutations within codons 2-7 of the mature GO coding sequence to enhance GO translation and have combined these with other expression-enhancing mutations. We selected the best E. coli host strain, autoinduction medium, induction temperature, harvest time and cell lysis procedure to produce active recombinant GO. Although normally secreted by the fungus, we have expressed GO in the cytoplasm of E. coli and have used a C-terminal Streptag II sequences for single-step affinity purification. This resulted in purification of 240 mg of functional GO per litre of shake flask culture. We have generated a saturation mutagenesis library at residue Cys383, known to influence substrate binding, and have used the optimised expression conditions to purify and characterise the resulting enzymes.     

Synthesis and evaluation of tetra(2,7-octadienyl) titanate as a reactive diluent for air-drying alkyd paints

Environmental regulations are forcing the reformulation of many decorative and protective coatings systems. In particular, air-drying solventborne alkyd paints need to meet increasingly stringent emission limits and often must be reformulated with suitable exempt solvents or reactive diluents to achieve volatile organic compound (VOC) reduction. In the research summarized in this article, a new reactive diluent, tetra(2,7-octadienyl) titanate, was synthesized and evaluated in alkyd formulations for VOC reduction and property enhancement. A soy-based long-oil alkyd resin; a soy-based high-solids, long-oil alkyd resin; and a linseed-based, high-solids long-oil alkyd resin were evaluated in combination with the new reactive diluent at weight percentages ranging from 10% to 50%. Characterization included measuring viscosity, film dry times, and film performance of the reactive diluent formulations in comparison to neat alkyd resins used as control. The tetra(2,7-octadienyl) titanate formulations consistently exhibited reduced viscosities and dry times as a function of concentration. The resulting films were harder and more thoroughly cured than the control systems.     

Supercooling of water droplets in jet aviation fuel

Ice formation in aircraft fuel systems is an ongoing problem with potentially disastrous consequences. Unfortunately, the icing of fuel systems is poorly understood. It is well known that at temperatures below 0 °C particles of H₂O suspended in fuel can exist as crystalline ice or metastable supercooled water. In this paper we show that micron sized water droplets immersed in Jet A-1 aviation fuel can exist in a metastable supercooled state to around −36 °C. In fact, the majority of droplets in our experiments froze homogeneously showing that the fuel itself did not catalyse ice formation. We suggest that H₂O particles will remain in a supercooled liquid state until they come into contact with a suitable solid surface in an aircraft’s fuel system or the temperature falls below the homogeneous freezing limit.     

Monomer sequence of partially hydrolyzed poly(4-tert-butoxystyrene) and morphology of diblock copolymers composing this polymer sequence as one block

The molecular characteristics of poly(4-tert-butoxystyrene) (O) upon hydrolysis reaction were investigated. It is known that O can be converted into poly(4-hydroxystyrene) (H) through hydrolysis reaction using strong acid. In this study, a set of poly(4-tert-butoxystyrene-co-4-hydroxystyrene)s (O/H copolymers) having various conversion rates, f_Hs, were prepared. Hydrolysis reaction is found to occur uniformly by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) where the average f_H obtained was consistent with that from ¹H NMR though a certain distribution in the number of hydrolyzed units was conceived. Monomer sequence of O/H copolymers was determined by ¹³C NMR and the changes in triad concentration were obtained by spectra subtraction method. As a result, ¹³C NMR reveals that O and H are statistically distributed. To evaluate the effect of hydrolysis on microphase-separated structure, we observed the morphology of partially hydrolyzed poly(4-tert-butylstyrene-block-4-tert-butoxystyrene) (BO) by transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS). Samples with f_H from 0.21 to 0.67 form both lamellar (major component) and cylindrical (minor component) structures reflecting both the statistical manner of hydrolysis reaction and the possible localized distribution of H sequence.     

Phylloquinone (vitamin K1) and dihydrophylloquinone content of fats and oils

Assessment of vitamin K (VK) dietary intakes has been limited by the incompleteness of VK food composition data for the U.S. food supply, particularly for VK-rich oils. The phylloquinone (VK-1) and 2′,3′-dihydrophylloquinone (dK) concentrations of margarines and spreads (n=43), butter (n=4), shortening (n=4), vegetable oils (n=6), and salad dressings (n=24) were determined by RP-HPLC with fluorescence detection. Each sample represented a composite of units or packages obtained from 12 or 24 outlets, which were geographically representative of the U.S. food supply. Butter, which is derived from animal fat sources, had less VK-1 compared to vegetable oil sources. The VK-1 and dK of the margarines and spreads increased with fat content and the degree of hydrogenation, respectively. In some margarines or spreads and in all shortenings, the dK concentrations were higher than the corresponding VK-1 concentrations. As the fat content of salad dressings increased, the VK-1 concentrations also increased. Fat-free foods had <1 μg/100 g of either form of the vitamin. No dK was detected in the salad dressings or oils tested. Some margarines, spreads, and salad dressings may be significant sources of vitamin K in the U.S. food supply.     

Dietary Supplementation of Calcium may Counteract Obesity in Mice Mediated by Changes in Plasma Fatty Acids

The scope of this study was to assess the impact of calcium and conjugated linoleic acid (CLA) supplementation on plasma fatty acid profiles and to evaluate potential synergistic effects of both compounds against dietary obesity. Mice separated into five experimental groups were followed: control (C), high-fat diet (HF), HF with calcium (Ca), HF plus CLA and HF with both Ca and CLA. Plasma metabolites and fatty acids were determined by commercial kits and gas chromatography, respectively. Both dietary calcium and CLA supplementation contributed to lower body fat gain under a HF diet. Maximum efficacy was seen with calcium; no additional effect was associated with the combined treatment with CLA. Plasma leptin, adiponectin and HOMA index were in accordance with an altered glucose/insulin homeostasis in the HF and HF + CLA groups, whereas control levels were attained under Ca-enriched diets. Plasma fatty acids showed minor changes associated to CLA treatment, but a high impact on PUFA was observed under Ca-enriched diets. Our results show that the mechanism underlying the anti-obesity effects of calcium supplementation is mediated mainly by changes in PUFA plasma profile. In addition, the lack of synergy on body weight reduction in combination with associated lipid profiles of calcium and CLA suggests that calcium may interfere with absorption and/or bioactivity of CLA, which can be of relevance when using CLA-fortified dairy products against human obesity.     

Synthesis and characterization of PVA/PES thin film composite nanofiltration membrane modified with TiO2 nanoparticles for better performance and surface properties

Novel polyethersulfone (PES)/poly (vinyl alcohol) (PVA)/titanium dioxide (TiO₂) composite nanofiltration membranes were prepared by dip-coating of PES membrane in PVA and TiO₂ nanoparticles aqueous solution. Glutaraldehyde (GA) was used as a cross-linker for the composite polymer membrane in order to enhance the chemical, thermal as well as mechanical stabilities. TiO₂ nanoparticles with different concentrations (0, 0.05, 0.1, 0.5 wt.%) were coated on the surface of PVA/PES composite membrane. The morphological study was investigated by atomic force microscopy (AFM), scanning surface microscopy (SEM) and along with X-ray diffraction (XRD). In addition, the membranes performances, in terms of permeate flux, ion rejection and swelling factor were also investigated. It was found that the increase in TiO₂ solution concentration can highly affect the surface morphology and filtration performance of coated membranes. The contact angle measurement and XRD studies indicated that the TiO₂ nanoparticles successfully were coated on the surface of PVA/PES composite membranes. However, rougher surface was obtained for membranes by TiO₂ coating. The filtration performance data showed that the 0.1 wt.% TiO₂-modified membrane presents higher performance in terms of flux and NaCl salt rejection. Finally, TiO₂ modified membranes demonstrated the lower degree of swelling.     

Glycosylation in Indolent,Significant and Aggressive Prostate Cancer by Automated High-Throughput N-Glycan Profiling

The diagnosis and treatment of prostate cancer (PCa) is a major health-care concern worldwide. This cancer can manifest itself in many distinct forms and the transition from clinically indolent PCa to the more invasive aggressive form remains poorly understood. It is now universally accepted that glycan expression patterns change with the cellular modifications that accompany the onset of tumorigenesis. The aim of this study was to investigate if differential glycosylation patterns could distinguish between indolent, significant, and aggressive PCa. Whole serum N-glycan profiling was carried out on 117 prostate cancer patients’ serum using our automated, high-throughput analysis platform for glycan-profiling which utilizes ultra-performance liquid chromatography (UPLC) to obtain high resolution separation of N-linked glycans released from the serum glycoproteins. We observed increases in hybrid, oligomannose, and biantennary digalactosylated monosialylated glycans (M5A1G1S1, M8, and A2G2S1), bisecting glycans (A2B, A2(6)BG1) and monoantennary glycans (A1), and decreases in triantennary trigalactosylated trisialylated glycans with and without core fucose (A3G3S3 and FA3G3S3) with PCa progression from indolent through significant and aggressive disease. These changes give us an insight into the disease pathogenesis and identify potential biomarkers for monitoring the PCa progression, however these need further confirmation studies.