Effect of atmospheric aging on volatility and reactive oxygen species of biodiesel exhaust nano-particles

In the prospect of limited energy resources and climate change, effects of alternative biofuels on primary emissions are being extensively studied. Our two recent studies have shown that biodiesel fuel composition has a significant impact on primary particulate matter emissions. It was also shown that particulate matter caused by biodiesels was substantially different from the emissions due to petroleum diesel. Emissions appeared to have higher oxidative potential with the increase in oxygen content and decrease of carbon chain length and unsaturation levels of fuel molecules. Overall, both studies concluded that chemical composition of biodiesel is more important than its physical properties in controlling exhaust particle emissions. This suggests that the atmospheric aging processes, including secondary organic aerosol formation, of emissions from different fuels will be different as well. In this study, measurements were conducted on a modern common-rail diesel engine. To get more information on realistic properties of tested biodiesel particulate matter once they are released into the atmosphere, particulate matter was exposed to atmospheric oxidants, ozone and ultra-violet light; and the change in their properties was monitored for different biodiesel blends. Upon the exposure to oxidative agents, the chemical composition of the exhaust changes. It triggers the cascade of photochemical reactions resulting in the partitioning of semi-volatile compounds between the gas and particulate phase. In most of the cases, aging lead to the increase in volatility and oxidative potential, and the increment of change was mainly dependent on the chemical composition of fuels as the leading cause for the amount and the type of semi-volatile compounds present in the exhaust.

A multi-criteria analysis approach for ranking and selection of microorganisms for the production of oils for biodiesel production

Oleaginous microorganisms have potential to be used to produce oils as alternative feedstock for biodiesel production. Microalgae (Chlorella protothecoides and Chlorella zofingiensis), yeasts (Cryptococcus albidus and Rhodotorula mucilaginosa), and fungi (Aspergillus oryzae and Mucor plumbeus) were investigated for their ability to produce oil from glucose, xylose and glycerol. Multi-criteria analysis (MCA) using analytic hierarchy process (AHP) and preference ranking organization method for the enrichment of evaluations (PROMETHEE) with graphical analysis for interactive aid (GAIA), was used to rank and select the preferred microorganisms for oil production for biodiesel application. This was based on a number of criteria viz., oil concentration, content, production rate and yield, substrate consumption rate, fatty acids composition, biomass harvesting and nutrient costs. PROMETHEE selected A. oryzae, M. plumbeus and R. mucilaginosa as the most prospective species for oil production. However, further analysis by GAIA Webs identified A. oryzae and M. plumbeus as the best performing microorganisms.

Purification of the major group 1 allergen from bahia grass pollen, Pas n 1

Background Group 1 grass pollen allergens are glycoproteins of the β-expansin family. They are a predominant component of pollen and are potent allergens with a high frequency of serum IgE reactivity in grass pollen-allergic patients. Bahia grass is distinct from temperate grasses and has a prolonged pollination period and wide distribution in warmer climates. Here we describe the purification of the group 1 pollen allergen, Pas n 1, from Bahia grass (Paspalum notatum), an important subtropical aeroallergen source. Methods Pas n 1 was purified from an aqueous Bahia grass pollen extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography, and assessed by one- and two-dimensional gel electrophoresis, immunoblotting and ELISA. Results Pas n 1 was purified to a single 29-kDa protein band containing two dominant isoforms detected by an allergen-specific monoclonal antibody and serum IgE of a Bahia grass pollen-allergic donor. The frequency of serum IgE reactivity with purified Pas n 1 in 51 Bahia grass pollen-allergic patients was 90.6%. Serum IgE reactivity with purified Pas n 1 was highly correlated with serum IgE reactivity with Bahia grass pollen extract and recombinant Pas n 1 (r = 0.821 and 0.913, respectively). Conclusions Pas n 1 is a major allergen reactive at high frequency with serum IgE of Bahia grass pollen-allergic patients. Purified natural Pas n 1 has utility for improved specific diagnosis and immunotherapy for Bahia grass pollen allergy.

A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 μg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

Advances in solid-catalytic and non-catalytic technologies for biodiesel production

The insecure supply of fossil fuel coerces the scientific society to keep a vision to boost investments in the renewable energy sector. Among the many renewable fuels currently available around the world, biodiesel offers an immediate impact in our energy. In fact, a huge interest in related research indicates a promising future for the biodiesel technology. Heterogeneous catalyzed production of biodiesel has emerged as a preferred route as it is environmentally benign needs no water washing and product separation is much easier. The number of well-defined catalyst complexes that are able to catalyze transesterification reactions efficiently has been significantly expanded in recent years. The activity of catalysts, specifically in application to solid acid/base catalyst in transesterification reaction depends on their structure, strength of basicity/acidity, surface area as well as the stability of catalyst. There are various process intensification technologies based on the use of alternate energy sources such as ultrasound and microwave. The latest advances in research and development related to biodiesel production is represented by non-catalytic supercritical method and focussed exclusively on these processes as forthcoming transesterification processes. The latest developments in this field featuring highly active catalyst complexes are outlined in this review. The knowledge of more extensive research on advances in biofuels will allow a deeper insight into the mechanism of these technologies toward meeting the critical energy challenges in future.

Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal beta 1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) crossreacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.

Use of a coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from Escherichia coli

We have designed a novel coupled transcriptional construct wherein Escherichia coil uracil DNA glycosylase (UDC:) and Bacillus subtilis phage PBS-2 encoded uracil DNA glycosylase inhibitor protein (Ugi) genes were cloned in tandem, downstream of an inducible promoter (P-trc). Use of this bicistronic operon has allowed purification of large amounts of UDG-Ugi complex formed in vivo. The system has also been exploited for purification of large amounts of Ugi. While establishing the expression system, one of the constructs showed detectable suppression of UAG termination codon and resulted in accumulation of a minor population of a putative readthrough polypeptide cor responding to UDG. We discuss the likely occurrence of such a phenomenon in overproduction of other recombinant proteins. Finally, the usefulness of the operon construct in convenient mutational analysis to study the mechanism of UDG-Ugi interaction is also discussed.

Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver

A number of studies in yeast have shown that DNA topoisomerase TI is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase rr in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-foe extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg2+-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Purification of pharmaceuticals and nutraceutical compounds by sub- and supercritical extraction and chromatography

This thesis discusses the use of sub- and supercritical fluids as the medium in extraction and chromatography. Super- and subcritical extraction was used to separate essential oils from herbal plant Angelica archangelica. The effect of extraction parameters was studied and sensory analyses of the extracts were done by an expert panel. The results of the sensory analyses were compared to the analytically determined contents of the extracts. Sub- and supercritical fluid chromatography (SFC) was used to separate and purify high-value pharmaceuticals. Chiral SFC was used to separate the enantiomers of racemic mixtures of pharmaceutical compounds. Very low (cryogenic) temperatures were applied to substantially enhance the separation efficiency of chiral SFC. The thermodynamic aspects affecting the resolving ability of chiral stationary phases are briefly reviewed. The process production rate which is a key factor in industrial chromatography was optimized by empirical multivariate methods. General linear model was used to optimize the separation of omega-3 fatty acid ethyl esters from esterized fish oil by using reversed-phase SFC. Chiral separation of racemic mixtures of guaifenesin and ferulic acid dimer ethyl ester was optimized by using response surface method with three variables per time. It was found that by optimizing four variables (temperature, load, flowate and modifier content) the production rate of the chiral resolution of racemic guaifenesin by cryogenic SFC could be increased severalfold compared to published results of similar application. A novel pressure-compensated design of industrial high pressure chromatographic column was introduced, using the technology developed in building the deep-sea submersibles (Mir 1 and 2). A demonstration SFC plant was built and the immunosuppressant drug cyclosporine A was purified to meet the requirements of US Pharmacopoeia. A smaller semi-pilot size column with similar design was used for cryogenic chiral separation of aromatase inhibitor Finrozole for use in its development phase 2.

Purification, properties and synthesis of delta-aminolaevulinate dehydratase from Neurospora crassa.

Delta-aminolaevulinate dehydratase, the second and rate-limiting enzyme of the haem-biosynthetic pathway, was purified 300-fold from induced cultures of Neurospora crassa. The native enzyme has a mol.wt. of about 350000, whereas the salt-treated enzyme after incubation at 37 degrees C for 10 min has a mol.wt. of about 232000. The mol.wt. of the subunit is about 38000. Antibodies to the purified enzyme were raised in rabbits. By using radiolabelling and immunoprecipitation techniques it was shown that addition of iron and laevulinate to iron-deficient cultures brings about a significant increase in the synthesis of the enzyme, and protoporphyrin, the penultimate end product of the pathway, represses enzyme synthesis.

Role of Protozoa in the Aerobic Purification of Sewage

DURING recent years, there has been increasing amount of evidence regarding the importance of protozoa in the aerobic purification of sewage1–4 and, more recently, some quantitative observations on protozoa in determining the condition of sludge and quality of effluent have been recorded5–10. The evidence so far obtained has, however, been only indirect, chiefly owing to the difficulty in separating the protozoa from the associated bacteria. This has now been achieved and the object of this note is to show that the isolated protozoa can bring about practically all the changes associated with the purification. The part played by the bacteria is almost negligible.

Enzymic Conversion of Agmatine to Putrescine inL athyrms satiuus seedlings - Purification and properties of a multifunctional enzyme (putrescine synthase)

The participation of a multifunctional enzy(am sein - gle polypeptide with multiple catalytic activities (14)) has been demonstrated in the conversion of agmatine to putrescine in Lathyrus sativus seedlings. This enzyme (putrescine synthase) with inherent activities of agmatine iminohydrolase, putrescine transcarbamylase, ornithine transcarbamylase, and carbamate has been purified to homogeneity anhda s M, = 55,000.

Purification of immunoglobulins from chicken sera by thiophilic gel chromatography

Immunoglobulin Y is different from most of the other immunoglobulins because it does not bind protein A or protein G. Thiophilic gel chromatography has been successfully used to purify IgY from chicken egg yolk, but the technology has not previously been used to purify IgY from serum. In this research note, we describe the optimization of T-gel chromatography for purification of IgY from serum. Data are provided on the recovery and purity of IgY obtained using potassium sulfate buffers of different concentrations. Decreasing the strength of potassium sulfate buffer from 0.5 to 0.3 M did not alter the amount of IgY recovered but increased the purity. Using 0.3 M potassium sulphate, we recovered approximately 63.7% of the serum Ig as almost pure IgY.

Purification of a bacterial organophosphate-hydrolysing phosphatase by cibacron 3GA-Sepharose affinity chromatography

A phosphatase catalysing the hydrolysis of organophosphorus pesticides was purified to homogeneity using Cibacron 3GA-Sepharose CL 6B affinity chromatography. The enzyme which is localized in the periplasm of the bacterium Image NC5 was extracted by treating with 0.2M MgCl2, pH 8.4. The enzyme was adsorbed to the Cibacron-Sepharose at pH 7.0 and eluted with Tris-HCl buffer at pH 8.0, with 47 per cent recovery. The enzyme thus obtained was electrophoretically homogeneous. This simple affinity purification procedure enhances the potential for its use in large scale detoxification systems.