Production of highly concentrated ethanol in a coupled fermentation/pervaporation process using silicalite membranes

The fermentation performance of a coupled fermentation/pervaporation process using silicalite membranes, which are ethanol permselective for an ethanol/water solution, was studied. The process exhibited about a 20% increase in an average glucose consumption rate as compared with that without the pervaporation unit. A strong correlation was observed between the membrane flux and the consumption rate. Ethanol concentrations in the permeates reached a maximum of 85% (v/v).     

Palladium complexes with hydrophosphorane ligands (HP∼O and HP∼N), catalysts for Heck cross-coupling reactions

The synthesis and structural characterization of palladium complexes 1-3 incorporating H-spirophosphorane HP(OCMe₂CMe₂O)₂ and H-phosphonate congener HP(O)(OCMe₂CMe₂O) ligands are reported. The chemical composition of the complexes [PdCl(μ-Cl){P(OCMe₂CMe₂O)OCMe₂CMe₂OH}]₂ (1), [Pd{P(O)(OCMe₂CMe₂O)}₂]_n (2), and [Pd(μ-Cl){P(OCMe₂CMe₂O)OH}{P(OCMe₂CMe₂O)O}]₂ (3) was established by means of NMR, IR, ESI-MS spectroscopic methods. The relative stereochemistry of 1 and 3 was unambiguously determined by single X-ray diffraction studies. It was proved that complexes 1-3, and complex [PdCl₂P(OCH₂CMe₂NH)OCH₂CMe₂NH₂] (4) previously described in the literature, are very efficient catalysts for the Heck cross-coupling of bromobenzene with n-butyl acrylate. Moreover, they were also found to be effective catalysts in the stereoselective synthesis of trans-stilbenes.     

Economic assessment of batch biodiesel production processes using homogeneous and heterogeneous alkali catalysts

An economic feasibility study on four batch processes for the production of biodiesel ranging from 1452 tonnes/year (5000 l/day) to 14,520 tonnes/year (50,000 l/day) is conducted. The four processes assessed are the (1) KOH-W process, characterized by a homogeneous KOH catalyst and hot water purification process; (2) KOH-D process, characterized by a homogeneous KOH catalyst and vacuum FAME distillation process; (3) CaO-W process, characterized by a heterogeneous CaO catalyst and hot water purification process; and (4) CaO-D process, characterized by a heterogeneous CaO catalyst and vacuum FAME distillation process. The costs of the waste cooking oil, fixed costs, and manufacturing costs for producing 7260 tonnes/year (25,000 l/day) of biodiesel by means of the four processes are estimated to be $248–256, $194–232, and $584–641 per tonne of biodiesel, respectively. Among the four processes, the manufacturing costs involved in the CaO-W process are the lowest, in the range from 1452 tonnes/year to 14,520 tonnes/year.     

Selective separation of tryptic beta-casein peptides through ultrafiltration membranes: Influence of ionic interactions

Peptide separation by selective membrane filtration has numerous potential applications such as production of peptides with biological activities or spectific enrichment in compounds acting as flavoring agents or as growth factors required by the fermentation industry. The retention of peptides arising from tryptic hdroysis of beta-casein using an M5 Carbosep membrane (molecular wieght cutoll = 10,000 D) has been studied. The peptides with known sequences were characterized by their molecular weight, isoelectric point, and hydrophobicity. Our experiments highlighted that their transmission involves mechanisms other than size exclusion as developed elsewhere. The effect of ionic interactions between peptides and membrance has been investgated by vrying pH, ionic strength of bulk, and electric potential of filtering material. The charge of both peptides and membrane plays an important role in the transmission, particularly with small size and high or lkow isoelectric point. Then, peptides with the same sign as the membrane have lower transmission than expected from the size xclusion model, whereas peptides with opposite sign have higher trnsmission than expected, and even higher than 1 with some of them. (c) 1995 John Wiley & Sons, Inc.     

Selective separation process of proteins based on the heat stress-induced translocation across phospholipid membranes

Heating of several protein solutions at 40–47°C for 5–60 min in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes induced the translocation of β-galactosidase (β-gal), α-glucosidase (α-glu) and bovine carbonic anhydrase (CAB) from outer to inner aqueous phase across the liposome membrane. The translocated amounts of β-gal at various temperatures were maximized under suitable heating conditions (45°C, 30 min). Those of α-glu and CAB were maximized at 40–45 and 60°C, respectively. Each maximum value could be correlated with the corresponding local hydrophobicity of each protein evaluated by the aqueous two-phase partitioning method. The possibility to apply these heat-induced translocation phenomena to the bioseparation of proteins was successfully demonstrated for the model mixture solution of β-gal, α-glu and CAB.     

Distribution of lectins in membranes of soybean and peanut plants

The distribution of lectin activity in soybean and peanut plants has been investigated. In both plants activity is found in all tissues examined (roots, shoots and leaves) at all stages of development from seedling to maturity (7 weeks). The cellular location of the lectins differs between soybean and peanut: in soybean the lectins are generally membrane-associated, whereas in peanut plants lectin activity is present also in the soluble cytoplasmic fraction. The membrane-associated lectins appear to differ from the seed lectins of the respective plants. The function of membrane-associated lectins is discussed.Abbreviations RCA lectin of castor bean - SBA soybean agglutinin - PNA peanut agglutinin - HEPES 2-[4-(2-Hydroxyethyl)-piperazinyl-(1)]ethanesulphonic acid - MES morpholinoethane sulphonic acid - PBS phosphate-buffered saline     

Rapid homogeneous lauroylation of wheat straw hemicelluloses under mild conditions

Hemicellulose-based hydrophobic biomaterials with degrees of substitution ranging from 0.46 to 1.54 were synthesized under mild conditions in homogeneous media (N,N-dimethylformamide-lithium chloride) by reacting the native wheat straw hemicellulosic polymers with lauroyl chloride using 4-dimethylaminopyridine as a catalyst. Other catalysts such as N-bromosuccinimide, N-methyl pyrrolidine, N-methyl pyrrolidinone, and pyridine were also investigated. Under optimum reaction conditions (2 equiv of lauroyl chloride and triethylamine per hydroxyl group, 5% 4-dimethylaminopyridine, 40 °C, 35 min), a high DS value of 1.54 was obtained. The biomaterials were characterized by FT-IR spectroscopy and ¹³C NMR spectroscopy as well as by thermal analysis. The results showed that the lauroylation occurred preferably at the C-3 hydroxyl group of β-d-Xylp units in the hemicelluloses, and the thermal stability of the hydrophobic polymers increased by esterification.     

Characterization of Four Amino Acids Constituting Cyl-2, a Metabolite from Cylindrocladium scoparium

A biologically active metabolite designated Cyl-2 from a phytopathogenic fungus, Cylindrocladium scoparium, was found to be composed of four amino acid residues, which fully account for all of the atoms constituting Cyl-2. These amino acids were identified as L-pipecolic acid, L-isoleucine, D-O-methyltyrosine and 2-amino-8-oxo-9,10-epoxydecanoic acid. Thus, Cyl-2 was established as a cyclotetrapeptide containing novel amino acid residues.     

Protein composition of Trypanosoma brucei mitochondrial membranes

Mitochondria consist of four compartments, outer membrane, intermembrane space, inner membrane, and matrix; each harboring specific functions and structures. In this study, we used LC‐MS/MS to characterize the protein composition of Trypanosoma brucei mitochondrial (mt) membranes, which were enriched by different biochemical fractionation techniques. The analyses identified 202 proteins that contain one or more transmembrane domain(s) and/or positive GRAVY scores. Of these, various criteria were used to assign 72 proteins to mt membranes with high confidence, and 106 with moderate‐to‐low confidence. The sub‐cellular localization of a selected subset of 13 membrane assigned proteins was confirmed by tagging and immunofluorescence analysis. While most proteins assigned to mt membrane have putative roles in metabolic, energy generating, and transport processes, ～50% have no known function. These studies result in a comprehensive profile of the composition and sub‐organellar location of proteins in the T. brucei mitochondrion thus, providing useful information on mt functions.     

Interaction of heat shock protein 70 with membranes depends on the lipid environment

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.     

Performance optimization of polysulfone ultrafiltration membranes for riboflavin separation using design experiments

Polysulfone membranes containing different concentrations of polyethylene glycol (M.Wt: 600 Da) as additive was prepared using N,N-dimethyl formamide as solvent. The parameters chosen for the study of separation of vitamin B₂ were concentrations of additive and feed and solvent evaporation time. Box-Behnken design methods and analysis of variance have been applied to the experimental separation of vitamin B₂ by polysulfone membranes. Box-Behnken design with three levels of additive concentration (2.5, 6.25 and 10 wt%), feed concentration (0.002, 0.005 and 0.008%) and solvent evaporation time (10, 20 and 30 s) is used for the identification of significant effects and interaction for the batch studied. Second order polynomial regression model which was used for analysis of the experiment made significant effect. The experimental values are in good correlation with predicted values, and the correlation coefficient was found to be 0.9194.     

Novel Method for the Covalent Immobilization of Oligonucleotides via Diels-Alder Bioconjugation

Abstract

The synthesis of cyclohexadiene and maleimide derivatives and their use for the functionalization of oligonucleotides and the coating of glass surfaces is reported. A method for the covalent attachment of diene or maleimide modified oligonucleotides to the coated glass surfaces via aqueous Diels-Alder reactions is presented.     

Synthesis and characterization of chitosan/ZnO nanoparticle composite membranes

Novel chitosan/ZnO nanoparticle (CS/nano-ZnO) composite membranes were prepared via the method of sol-cast transformation and studied by UV-vis absorption spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive X-ray fluorescence spectrometry (EDX). The characterization revealed that ZnO nanoparticles dispersed homogeneously within the chitosan matrix. The mechanical and antibacterial properties of the product were investigated. The results showed that the ZnO content had an effect on the mechanical properties of CS/nano-ZnO composite membranes, and that the antibacterial activities of CS membranes for Bacillus subtilis, Escherichia coli, and Staphylococcus aureus were enhanced by the incorporation of ZnO. Further, CS/nano-ZnO composite membranes with 6-10 wt % ZnO exhibited high antibacterial activities.     

Plasma membrane lipid diffusion and composition of sea urchin egg membranes vary with ocean temperature

A diverse and complex array of lipids plays a vital role in structuring and organizing cell membranes. However, the details of lipid requirements for global membrane organization are poorly understood. One obstacle to this understanding is the difficulty of accurately manipulating the lipid composition of commonly studied mammalian cells. In contrast, the lipid composition of cells of ectotherms changes with changes in environmental temperatures. Thus, comparison of lipid probe diffusion in cells from animals living at different temperatures, together with biochemical analysis, can be used toward understanding membrane organization. We used two dialkyindocarbocyanine iodide (DiI) probes, of differing chain length, to probe lipid organization in terms of their lateral diffusion in eggs of the sea urchin Strongylocentrotus purpuratus. The lateral diffusion of our probes changed in urchins developing in the year of an "El Ni?o" weather event, which raised the ocean temperature by several degrees, suggesting alterations in membrane domain composition and structure. Indeed the changes in lateral diffusion were correlated with lower levels of unsaturated fatty acids and cholesterol in animals of the "El Ni?o" year than in animals of the preceding or following years. We found similar trends comparing DiI diffusion in membranes of eggs from 15 degrees C waters with those from 10 degrees C. Our findings establish a new approach for manipulating and studying membrane organization.     

Selective precipitation and recovery of xylanase using surfactant and organic solvent

The selective precipitation of xylanase from an aqueous phase containing mixtures of xylanase and cellulase was studied using an ionic surfactant as precipitating ligand and a polar organic solvent as recovery solvent. Of four ionic surfactants tested, sodium di-(2-ethylhexyl) sulfosuccinate (AOT) showed a complete removal of xylanase at pH 4.5. The recovery of xylanase from the xylanase-AOT complex was a strong function of the type and the volume of the polar solvent and of the concentration of sodium acetate buffer in the final aqueous solution used to solubilize the recovered xylanase. With ethanol as a recovery solvent, a recovery of xylanase activity of 78 +/- 10% was obtained. The cellulase activity in the recovered xylanase was below the detection limit. The results demonstrate that an ionic surfactant can recover enzymes from aqueous solutions without loss in their activity.     

Hydrolysis of the amide bond in methionine-containing peptides catalyzed by various palladium(II) complexes: Dependence of the hydrolysis rate on the steric bulk of the catalyst

¹H NMR spectroscopy was applied to study the reactions of cis-[Pd(L)(H₂O)₂]²⁺ complexes (L is en, pic and dpa) with the N-acetylated tripeptides L-methionylglycylglycine, MeCOMet–Gly–Gly, and glycyl–L-methionyl–glycine, MeCOGly–Met–Gly. All reactions were performed in the pH range 2.0–2.5 with equimolar amounts of the cis-[Pd(L)(H₂O)₂]²⁺ complex and the tripeptide at 60 °C. The hydrolytic reactions of the cis-[Pd(en)(H₂O)₂]²⁺, cis-[Pd(pic)(H₂O)₂]²⁺ and cis-[Pd(dpa)(H₂O)₂]²⁺ complexes with MeCOMet–Gly–Gly were regioselective and only the amide bond involving the carboxylic group of methionine was cleaved. However, in the reactions of these three Pd(II) complexes with MeCOGly–Met–Gly, two amide bonds, Met–Gly and MeCO–Gly, were cleaved. From UV–Vis spectrophotometry studies, it was found that the rate-determining step of these hydrolytic reactions is the monodentate coordination of the corresponding Pd(II) complex to the sulfur atom of the methionine side chain. The rate of the cleavage of these amide bonds is dependent on the nature of the bidentate coordinated diamine ligand L (en > pic > dpa). The hydrolytic reaction of cis-[Pd(L)(H₂O)₂]²⁺-type complexes with MeCOMet–Gly–Gly, containing the methionine side chain in the terminal position of the peptide, is regioselective while in the reaction of these Pd(II) complexes with MeCOGly–Met–Gly, none selective cleavage of the peptide occurs. This study contributes to a better understanding of the selective cleavage of methionine-containing peptides employing palladium(II) complexes as catalysts.     

Flicker noise of ion-selective membranes and turbulent convection in the depleted layer

Flicker noise of electric currents through ion-selective membranes is explained. It is attributed to the depletion of salt on one side of the membrane, which creates a thin layer of high resistance. Joule heating in this depletion layer and the ensuing temperature gradient, as well as the concentration gradient, give rise to buoyant forces which may create a turbulent convection current. The turbulence mixes the depletion layer so that the electric resistance fluctuates, and consequently the current flickers.Experiments with ion-selective membranes support this conjecture. They show that 1) Noise is coincident with the increase of the electric resistance by the depletion process. 2) When the current density is reduced, it reaches a critical value, below which the convection current changes from turbulent to laminar, and the noise disappears. 3) Noise reduces with temperature, because the expansion coefficient of water decreases with temperature, and its viscosity increases. 4) A non-ionic water-soluble polymer added to the compartment on the side of the depletion layer reduces the noise, by increasing the bulk viscosity of the solution. 5) Noise depends on the membrane's orientation in the gravitational field. 6) The convection-current in the depletion layer can be observed directly, using a laser-beam, by adding latex particles which create optical noise as they drift with the convection current across the beam. The optical noise is observed only coincidently with the current noise.A. Katzir-Katchalsky Fellow     

Solid-state NMR study of antimicrobial peptides from Australian frogs in phospholipid membranes

Antimicrobial peptides, isolated from the dorsal glands of Australian tree frogs, possess a wide spectrum of biological activity and some are specific to certain pathogens. These peptides have the capability of disrupting bacterial membranes and lysing lipid bilayers. This study focused on the following amphibian peptides: (1) aurein 1.2, a 13-residue peptide; (2) citropin 1.1, with 16 residues; and (3) maculatin 1.1, with 21 residues. The antibiotic activity and structure of these peptides have been studied and compared and possible mechanisms by which the peptides lyse bacterial membrane cells have been proposed. The peptides adopt amphipathic -helical structures in the presence of lipid micelles and vesicles. Specifically ¹⁵N-labelled peptides were studied using solid-state NMR to determine their structure and orientation in model lipid bilayers. The effect of these peptides on phospholipid membranes was determined by ²H and ³¹P solid-state NMR techniques in order to understand the mechanisms by which they exert their biological effects that lead to the disruption of the bacterial cell membrane. Aurein 1.2 and citropin 1.1 are too short to span the membrane bilayer while the longer maculatin 1.1, which may be flexible due to the central proline, would be able to span the bilayer as a transmembrane -helix. All three peptides had a peripheral interaction with phosphatidylcholine bilayers and appear to be located in the aqueous region of the membrane bilayer. It is proposed that these antimicrobial peptides have a detergent-like mechanism of membrane lysis.This paper was submitted as a record of the 2002 Australian Biophysical Society