Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six differentlinker peptides to join the V_H and V_L domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)₃ linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcore^TM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric.     

Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment

Europium chelates provide a non-radioactive alternative forsensitive labelling of antibodies for diagnostic immunoassays.Lysine residues at antibody surfaces are ready targets for labellingby an isothiocyanate derivative of the europium chelate (Eu³⁺).Here the labelling efficiency of a recombinant anti-human -fetoprotein(hAFP) Fab fragment has been improved by increasing its lysinecontent by protein engineering. Molecular modelling was usedto identify three light chain constant domain surface arginineresidues, R154, R187 and R210, which were mutated to lysineresidues. The mutations did not influence the affinity of thelysine-enriched Fab fragment and its labelling efficiency wasfound to be 40% higher than that of the wildtype Fab fragmentWith low degree of labelling, the affinities of the two Fabfragments were identical and comparable with that of the originalmonoclonal anti-hAFP IgG. With a higher degree of labellingthe affinities of both Fab fragments decreased more than thatof the intact IgG since more lysine residues are available forlabelling in the additional heavy chain constant domains ofthe larger molecule. Electrostatic adsorption and covalent immobilizationof the Fab fragments were characterized by BIAcore^TM and thelysine-enriched Fab fragment was found to be more efficientlyimmobilized to an activated carboxymethyl surface.     

Volatile monocarbonyl compounds of carrot roots at various stages of maturity

Summary Volatile carbonyl compounds of carrot varietyFeonia Hunderup S-64 at various stages of maturity were analysed as their 2,4-DNPH's by a combined TLC-GLC-MS method. Twenty-three different carbonyl compounds were identified, of which undecanal, buten-2-al, methylbutenal, pentan-2-one, 6-methyl-5-hepten-2-one and 5-methylfurfural have not previously been found in carrot. During maturation the content of acetaldehyde and acetone increased significantly, while that of methylbutenal decreased.

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Die leichtflüchtigen Monocarbonylverbindungen der Karotten in verschiedenen Stufen der Reife

Zusammenfassung Die leichtflüchtigen Carbonylverbindungen der Karotten Varietät Feonia Hunderup S-64 wurden in verschiedenen Stufen der Reife als 2,4-DNPH mit der kombinierten DC-GC-MS-Methode analysiert. Dreiundzwanzig verschiedene Carbonylverbindungen wurden nachgewiesen. Von diesen sind Undecanal, 2-Butenal, Methylbutenal, 2-Pentanon, 6-Methyl-5-hepten-2-on und 5-Methylfurfural noch nicht in Karotten aufgefunden worden. Während des Reifens nahm der Gehalt an Acetaldehyd und Aceton bedeutend zu. Der Gehalt an Methylbutenal verminderte sich.

     

Changes in soil carbon stock after cropland conversion to grassland in Russian temperate zone: measurements versus model simulation

The collapse of Soviet Union in early 1990s led to abandonment of large area of arable land which is assumed to act as a carbon (C) sink. We studied the ability of two dynamic soil C models (Yasso07 and RothC) to predict changes in soil C content after cropland abandonment. The performance of the models was compared using the results of a long-term experiment in Pushchino, Moscow region (54°50′N, 37°35′E) in Russia. The experiment was divided in four combinations of fertilizer or mowing treatments on former cropland soil. The soil C content was determined in the year of establishment (1980) and thereafter in 1999 and 2004. The soil C stocks increased by about 1.5- to 1.8-fold during the study period. Both models predicted the overall change in soil C relatively well (modelling efficiency of Yasso07 and RothC were 0.60 and 0.73, respectively). According to the models, the soil gained on average 140–150 g C m^?2 year^?1 during the first 5 years after conversion of cropland to grassland. The C sequestration rate decreased to 40–50 g C m^?2 year^?1 after 20 years of land use change. The sequestration rates estimated in this study are comparable to the rates observed in other studies.     

Enzyme Processing of Soy Flour with Minimized Protein Loss

The enzymatic treatment of defatted soy flour (SF) to reduce indigestible carbohydrates can result in undesirable protein loss. Here protein loss was minimized with quantitation of the effects of ionic strength (IS), protease activity, and SF toasting. At the enzyme processing condition (25% w/v SF, 50 °C, pH 4.8, 48 hours), protein loss increased linearly with the IS in enzyme broths (EB); e.g., contacting untoasted SF with water or heat-deactivated EB showed protein loss of 28% in water but up to 40% when IS was increased in the range of 0.04–0.19 M. Protein loss also increased with protease in EB (nondeactivated): after adjusted for IS-related loss, approximately 10% and 25% additional protein loss occurred in EB of 73 and 490–557 U/(g SF) protease, respectively. SDS-PAGE results showed that proteolysis was not extensive, mainly on β-conglycinin α'/α and glycinin acidic 37-kDa subunits; and most of the proteolytic products could be recovered by heat-induced precipitation. SF toasting effects were studied, particularly at 2-hours 160°C, with material balances, protein distributions, and monosaccharide yields in hydrolysates. Overall, protein loss was minimized to 5.2% and the conversion of carbohydrate to monomeric sugars increased to 89.2%.     

Optimization of Enzymatic Process Condition for Protein Enrichment,Sugar Recovery and Digestibility Improvement of Soy Flour

Soy protein is a valuable nutritional supplement for food and animal feed. While protein constitutes ~50 % of defatted soy flour (SF), it coexists with complex carbohydrates (30–35 %) which may have anti‐nutritional effects. An enzymatic process can remove the carbohydrate and produce protein‐enriched soy products. The hydrolysate with monomerized carbohydrates is valuable fermentation feedstock. In this study, Aspergillus niger and Trichoderma reesei enzymes were compared for use in the process. Effects of pH (3.2–6.4), temperature (40–60 °C), enzyme‐to‐SF ratio (0–2 ml/g) and SF loading (150–350 g/l) were evaluated for the enzymatic conversion of SF carbohydrate to reducing sugar (Y_RS) and total soluble carbohydrate (Y_TC) in the hydrolysate. Effects of these single factors and interactions between factors were investigated. Optimal pH and temperature were similar for both enzymes: pH 4.8 and 50–51 °C for Y_TC, and pH 5.1–5.2 and 48–51 °C for Y_RS. The two enzymes also gave similar protein contents in resultant soy protein concentrates, i.e., 74–75 % with 2 ml/g enzyme broth and 150 g/l SF, which were higher than the 64–68 % protein in commercial concentrates. A. niger enzyme was significantly more effective in carbohydrate conversion, achieving Y_RS = 75 % and Y_TC = 78 % with 2 ml/g enzyme and 150 g/l SF, higher than the Y_RS (30 %) and Y_TC (64 %) obtained with T. reesei enzyme. Monomerization was essentially complete in hydrolysate produced with A. niger enzyme.     

Impact toughness,viscoelastic behavior,and morphology of polypropylene–jute–viscose hybrid composites

In this investigation, we studied the impact toughness and viscoelastic behavior of polypropylene (PP)–jute composites. In this study, we used viscose fiber as an impact modifier and maleated PP as a compatibilizer. The toughness of the composites was studied with conventional Charpy and instrumental falling‐weight impact tests. The composites’ viscoelastic properties were studied with dynamic mechanical analysis. The results show that the incorporation of viscose fibers improved the impact strength and toughness to 134 and 65% compared to those of the PP–jute composites. The tan δ peak amplitude also increased with the addition of the impact modifier and indicated a greater degree of molecular mobility. The thermal stability of the composites was evaluated with thermogravimetric analysis. The addition of 2 wt % maleated polypropylene (MAPP) to the impact‐modified composite improved the impact strength and toughness to 144 and 93%, respectively. The fiber–matrix morphology of the fracture surface and the Fourier transform infrared spectra were also studied to ascertain the existence of the type of interfacial bonds. Microstructural analysis showed the retention of viscose fibers in the composites compared to the more separated jute fibers. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 42981.     

Bio-methanol: How energy choices in the western United States can help mitigate global climate change

Converting available biomass from municipal, agricultural and forest wastes to bio-methanol can result in significant environmental and economic benefits. Keeping these benefits in mind, one plausible scenario discussed here is the potential to produce energy using bio-methanol in five states of the western United States. In this scenario, the bio-methanol produced is from different biomass sources and used as a substitute for fossil fuels in energy production. In the U.S. West, forest materials are the dominant biomass waste source in Idaho, Montana, Oregon and Washington, while in California, the greatest amount of available biomass is from municipal wastes. Using a 100% rate of substitution, bio-methanol produced from these sources can replace an amount equivalent to most or all of the gasoline consumed by motor vehicles in each state. In contrast, when bio-methanol powered fuel cells are used to produce electricity, it is possible to generate 12–25% of the total electricity consumed annually in these five states.As a gasoline substitute, bio-methanol can optimally reduce vehicle C emissions by 2–29 Tg of C (23–81% of the total emitted by each state). Alternatively, if bio-methanol supported fuel cells are used to generate electricity, from 2 to 32 Tg of C emissions can be avoided. The emissions avoided, in this case, could equate to 25–32% of the total emissions produced by these particular western states when fossil fuels are used to generate electricity. The actual C emissions avoided will be lower than the estimates here because C emissions from the methanol production processes are not included; however, such emissions are expected to be relatively low. In general, there is less carbon emitted when bio-methanol is used to generate electricity with fuel cells than when it is used as a motor vehicle fuel.In the state of Washington, thinning “high-fire-risk” small stems, namely 5.1–22.9 cm diameter trees, from wildfire-prone forests and using them to produce methanol for electricity generation with fuel cells would avoid C emissions of 3.7–7.3 Mg C/ha. Alternatively, when wood-methanol produced from the high-fire-risk wood is used as a gasoline substitute, 3.3–6.6 Mg C/ha of carbon emissions are avoided. If these same “high-fire-risk” woody stems were burned during a wildfire 7.9 Mg C/ha would be emitted in the state of Washington alone. Although detailed economic analyses of producing methanol from biomass are in its infancy, we believe that converting biomass into methanol and substituting it for fossil-fuel-based energy production is a viable option in locations that have high biomass availability.