An ultrafiltration membrane bioreactor for the lipolysis of olive oil in reversed micellar media

The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of dioctyl sodium sulfosuccinate (AOT) in isooctane was investigated in an ultrafiltration ceramic membrane reactor of tubular type, operating in a batch mode. Water concentration was found to be a critical parameter in the enzyme kinetics and hydrolysis yield of the reaction. The size of micelles, recirculation rate, and substrate concentration were found to be the major factors affecting the separation process. A correlation that enables the prediction of final conversion degrees in this bioreactor from the initial reaction conditions was established. (c) 1993 Wiley & Sons, Inc.     

Separation and purification of periplasmic cytochrome c553 using reversed micelles

Surfactant concentration, ionic strength, and pH were optimised for the selective separation and purification of periplasmic cytochrome c553 from recombinant E. coli TG2 cells using response surface methodology. Back-extraction was accomplished using counter-ionic surfactant addition. Optimum forward extraction conditions were: 65 mM bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT), 0.07 M NaCl, and pH 8.4, while the optimum back-extraction conditions were 80 mM trioctylmethylammonium chloride, 0.85 M KCl, and pH 9.62. In comparison to a conventionally purified sample using column chromatography (10 mg cytochrome c553 l–1 with a purity of 0.66), reverse micelles achieved the same concentration and similar purity (0.50) in only two simple steps.     

Performance of a cutinase membrane reactor for the production of biodiesel in organic media

The enzymatic transesterification of oils with an alcohol, using recombinant cutinase of Fusarium solani pisi microencapsulated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane reversed micelles, was performed in a membrane bioreactor (MBR). A tubular ceramic membrane with a nominal molecular weight cut off of 15,000 Da was used to retain the enzyme, and characterized in terms of rejection coefficients of the reaction components by transmission experiments. The performance of the MBR in a total recirculation-batch mode was compared with results obtained in a stirred batch tank reactor. The continuous operation of the MBR was also evaluated and the influence of the alcohol type and permeate flow rate on conversion degree and productivity (up to 500 g(product) /day/g(enzyme) was attained) were analyzed. Cutinase wild type and mutant T179C were tested for this process and the high long-term operational stability of the cutinase mutant demonstrated its potential as biocatalyst for the enzymatic continuous production of biodiesel.     

Solubilization of enzymes in water-in-oil microemulsions and their rapid and efficient release through use of a pH-degradable surfactant

α-Chymotrypsin and lysozyme were solubilized in a water/O-[(2-tridecyl, 2-ethyl-1,3-dioxolan-4-yl)methoxy]–O′-methoxy poly(ethylene glycol) (CK-2,13 surfactant)/isooctane water-in-oil microemulsion solution at 1.5–2 and 10 g l⁻¹ for 0.15 and 1.2 M CK-2,13, respectively. Upon contact with an equal volume of 0.1 M NaH₂PO₄/Na₂HPO₄ buffer, pH 5, a three-phase system (Winsor-III system) was formed, consisting of a surfactant-rich middle phase and aqueous and isooctane-rich “excess” phases. Both enzymes were rapidly released into the aqueous excess phase, with 70% recovery of each in 30 and 60 min for microemulsion solutions containing 0.15 and 1.2 M surfactant, respectively. The recovered enzymes retained >90% of their original specific activity.     

Nanoscale amphiphilic macromolecules as lipoprotein inhibitors: the role of charge and architecture

A series of novel amphiphilic macromolecules composed of alkyl chains as the hydrophobic block and poly(ethylene glycol) as the hydrophilic block were designed to inhibit highly oxidized low density lipoprotein (hoxLDL) uptake by synthesizing macromolecules with negatively charged moieties (ie, carboxylic acids) located in the two different blocks. The macromolecules have molecular weights around 5,500 g/mol, form micelles in aqueous solution with an average size of 20-35 nm, and display critical micelle concentration values as low as 10(-7) M. Their charge densities and hydrodynamic size in physiological buffer solutions correlated with the hydrophobic/ hydrophilic block location and quantity of the carboxylate groups. Generally, carboxylate groups located in the hydrophobic block destabilize micelle formation more than carboxylate groups in the hydrophilic block. Although all amphiphilic macromolecules inhibited unregulated uptake of hoxLDL by macrophages, inhibition efficiency was influenced by the quantity and location of the negatively charged-carboxylate on the macromolecules. Notably, negative charge is not the sole factor in reducing hoxLDL uptake. The combination of smaller size, micellar stability and charge density is critical for inhibiting hoxLDL uptake by macrophages.     

Determination of helical membrane protein topology using residual dipolar couplings and exhaustive search algorithm: application to phospholamban

Dipolar waves are distinct hallmarks of both the secondary and tertiary structures of alpha-helical proteins that are immobilized in membrane bilayers or embedded in anisotropic media. We present a simple, semi-empirical approach that exploits the modulation of the amplitude and average of dipolar waves to determine the topology of alpha-helical proteins. Moreover, we describe the application of this method for the structural determination of a detergent solubilized membrane protein, phospholamban (PLB) that is involved in calcium regulation of cardiac muscle. When combined with high-resolution solid-state NMR data, this method can serve as a fast route for determining the topology of helical membrane proteins solubilized in detergent micelles.     

Simulation of the N-terminus of HIV-1 glycoprotein 41000 fusion peptide in micelles.

In this paper, the N-terminus of glycoprotein-41, the HIV-1 fusion peptide, was studied by molecular dynamics simulations in an explicit sodium dodecyl sulfate micelle. The simulation provides a detailed picture of the equilibrium structure and peptide stability as it interacts with the micelle. The equilibrium location of the peptide shows the peptide at the surface of the micelle with hydrophobic residues interacting with the micelle's core. At equilibrium, the peptide adopts an alpha-helical structure from residues 5-16 and a type-1 beta-turn from 17-20 with the other residues exhibiting more flexible conformations. The primary hydrophobic interactions with the micelle are from the leucine and phenylalanine residues (Leu-7, Phe-8, Leu-9, Phe-11, Leu-12) while the alanine and glycine residues (Ala-1, Gly-3, Gly-5, Ala-6, Gly-10, Gly-13, Ala-14, Ala-15, Gly-16, Gly-10, Ala-21) interact favorably with water molecules. The results suggest that Phe-8, part of the highly conserved FLG motif of the fusion peptide, plays a key role in the interaction of the peptide with membranes. Our simulations corroborate experimental investigations of the fusion peptide in SDS micelles, providing a high-resolution picture that explains the experimental findings.     

Trypsin In Lecithin Based w/o Microemulsions. Fluorescence and Enzyme Activity Studies

Lecithin based microemulsions were used as model systems for enzymic studies. The phase behavior of the system: purified soya bean lecithin/propan-1-ol/isooctane/water was examined. It was found that the ability of the system to solubilize water was strongly affected by the lecithin and alcohol concentrations. Trypsin was entrapped in lecithin microemulsion systems of different composition and tested for proteolytic activity on the hydrolysis of lysine-p-nitroanilide (LNA). The kinetic constants were determined and in most cases the ratio k_cat/Km was higher than that observed in aqueous solution. The optimum enzyme activity was found at pH 9 for the system formulated with 5% w/w lecithin in isooctane, while increasing w_o, where w_o = [H₂o]/[Lecithin], the enzyme activity followed a bell-shaped pattern with a maximum at w_o= 20. The stability of trypsin in microemulsions was higher in the low water containing systems. Using the fluorescence quenching technique it was found that the system compartmentalization depended on the water content and the presence of the enzyme. Time-resolved luminescence decay studies were carried out to clarify the effect of the water content and the presence of the enzyme molecules on the micro-emulsion structure. The analysis of the luminescence data was done with a “percolation” model of stretched exponential. A dramatic variation of the water/oil interface occurred above the percolation threshold, while the addition of the enzyme induced a more restricted microenvironment.     

Ultrastructure and development during meiosis and the tetrad period of sporogenesis in the leptosporangiate fern Alsophila setosa (Cyatheaceae) compared with corresponding stages in Psilotum nudum (Psilotaceae)

The pre-meiotic, meiotic and tetrad stages of development in microsporangia of Alsophila setosa were studied with particular emphasis on the early establishment of patterning in the microspore wall and the subsequent development of the sporoderm. The data obtained were compared with corresponding ontogenetic stages of Psilotum nudum. Tapetal behaviour was also examined. During the tetrad period, only one layer, a thin undulating sheet, appeared alongside the plasma membrane of the tetraspores, and this was evidently formed on a pre-patterned structure – a fibrillar layer, corresponding to a kind of primexine matrix. The early free microspores had a wavy plasma membrane with a parallel, sinusoidal, thin initial sporoderm layer. The proximal apertural fold was observed to be an extended outgrowth of this initial spore envelope. Sporoderm ontogeny during the tetrad period in Alsophila and Psilotum show some common points, but also fundamental differences, mainly in the relative timing of events: in Alsophila the end of the tetrad period is the starting point for exospore development, whereas in Psilotum the exospore is already complete at this stage. Considerable differences were also observed in the tapetum of the two species.     

Micellar environments induce structuring of the N-terminal tail of the prion protein

In the physiological form, the prion protein is a glycoprotein tethered to the cell surface via a C-terminal glycosylphosphatidylinositol anchor, consisting of a largely alpha-helical globular C-terminal domain and an unstructured N-terminal portion. This unstructured part of the protein contains four successive octapeptide repeats, which were shown to bind up to four Cu(2+) ions in a cooperative manner. To mimic the location of the protein on the cell membrane and to analyze possible structuring effects of the lipid/water interface, the conformational preferences of a single octapeptide repeat and its tetrameric form, as well of the fragment 92-113, proposed as an additional copper binding site, were comparatively analyzed in aqueous and dodecylphosphocholine micellar solution as a membrane mimetic. While for the downstream fragment 92-113 no conformational effects were detectable in the presence of DPC micelles by CD and NMR, both the single octapeptide repeat and, in an even more pronounced manner, its tetrameric form are restricted into well-defined conformations. Because of the repetitive character of the rigid structural subdomain in the tetrarepeat molecule, the spatial arrangement of these identical motifs could not be resolved by NMR analysis. However, the polyvalent nature of the repetitive subunits leads to a remarkably enhanced interaction with the micelles, which is not detectably affected by copper complexation. These results strongly suggest interactions of the cellular form of PrP (PrP(c)) N-terminal tail with the cell membrane surface at least in the octapeptide repeat region with preorganization of these sequence portions for copper complexation. There are sufficient experimental facts known that support a physiological role of copper complexation by the octapeptide repeat region of PrP(c) such as a copper-buffering role of the PrP(c) protein on the extracellular surface.