Reverse micelles as a bioseparation tool

Most of the recent studies in biotechnology have focused on upstream developments such as genetic engineering and bioreactor design. In contrast, downstream processing, which is a major part of production costs, has received disproportionately little attention. Development of techniques that can be scaled up into feasible downstream technology is needed. Selective solubilization of proteins and other biomolecules in reverse micellar organic phase is a promising technique that has the potential to be developed into a liquid-liquid extraction technology for bioseparation. This review describes the concept of ‘enzymes in reverse micelles’ and discusses the ways in which various parameters govern protein solubilization in reverse micelles. Also, a general rationale is provided for developing a process for bioseparation by exploiting the reverse micelle phenomenon.     

Reverse micelles as reaction media for lipases

Reversed micelles are at the present time faced as common organic media to perform biocatalysis. They have been associated to the idea of a microreactor where the enzyme can be sheltered and protected from solvent detrimental effects. This simplistic idea led some investigators to ignore some basic understanding, such as the recognition of the enzyme-specific microenvironment and what the enzyme experiences inside the reversed micelle. To date the number of reactions catalyzed by lipases in reversed micelles is large. This review aims to highlight some of the fundamental aspects of the lipase microencapsulation as well as to resume the outstanding progress of the reversed micellar systems. The properties of the micellar microenvironment are reviewed and related to the lipases' performance both in terms of activity and stability. The heterogeneity of reversed micellar systems is discussed in relation to component distribution models and also to enzymatic kinetics. The new trends and the practical aspects where efforts should be centralized in order to spread out the micellar bioreactor technology over industrial processes are also discussed.     

Reverse micelles as a versatile medium for the study of lactate dehydrogenase in vitro

Rabbit muscle lactate dehydrogenase has been solubilized in cationic reverse micelles of cetyltrimethyl ammonium bromide (CTAB) and isooctane-chloroform (1:1, V/V). The activity of the enzyme was notably affected by the change in water pool, pH, and concentration of the surfactant. Lactate dehydrogenase showed its full activity in this reverse micellar system in non-aqueous solvent under specific conditions at a Wo value of 30.55, pH 7.0, and 100 mM CTAB in comparison to the activity measured in aqueous system under optimum conditions. These results indicate that even the large and complex enzymes (M.W. hundred thousand and four subunits) can be solubilized in apolar solvents where they may retain their conformational integrity and oligomericity, i.e., optimum subunit-subunit interaction with maintenance of full activity.     

Reverse micelles,a system for antibody-catalysed reactions

This work presents some aspects of the application of catalytic antibodies in water-in-oil microemulsions (reverse micelles) based on sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) in isooctane. The monoclonal antibody (mAb) 9A8 used in this study is a fully characterised acetylcholinesterase-like antibody produced by the anti-idiotypic approach. The effect of various parameters, such as, the size and the concentration of reverse micelles, as well as the concentration and the nature of substrates on abzyme catalytic activity were investigated.     

Reverse micelles: a novel tool for H2 production

Reverse micelles serve as a novel tool to entrap enzymes and microbial whole cells within aqueous pockets and can be of great use in enhancing the efficiency and sustainability of the biological system. Photosynthetic bacterium Rhodopseudomonas sphaeroides entrapped inside the aqueous pool of reverse micelles prepared from benzene-sodium lauryl sulphate exhibited 25-fold enhancement of H₂ photoproduction rate (1.67 ml H₂ [mg protein]^–1 h^–1) compared to cells suspended in normal aqueous medium. Hydrogen photoproduction by the bacterium was catalysed by the nitrogenase enzyme system which was supported at a low light intensity of 12 Em^–2 sec^–1 photon flux energy at a wavelength of 520 nm. The optimum temperature for the process was 40 °C.     

Surfactant micelles containing protoporphyrin IX as models of primitive photocatalytic systems: a spectral study

Micelles of various surfactants containing protoporphyrin IX (PPIX) have been studied as models of primitive membrane-like photocatalytic systems. Spectral characteristics (absorption spectra, fluorescence emission and excitation spectra, fluorescence quantum yields and lifetimes) have been measured to indicate specific interactions of PPIX molecules with the micelles. Two types of PPIX aggregates are probably formed in water: non-fluorescent clusters corresponding to the absorption peak at 642-648 nm and fluorescent friable dimers with strong solute-solvent interactions corresponding to the absorption peak at 618 nm. The aggregates are solubilized by the micelles, which results in an increase of the fluorescence quantum yield (and thus in the increase of the PPIX sensitizing ability). Solubilization of PPIX molecules by SDS-micelles is enhanced by the partial neutralization of the negative surface charge of the micelles. Neutral Triton X-100 micelles solubilize PPIX much better than SDS particles; however, some of the clusters formed in the bulk aqueous phase of the detergent-water system remain aggregated in the crown of the micelle. The positively charged CTAB micelles have been shown to provide the best solubilization of PPIX accompanied by the highest increase of its emitting activity. The results are discussed in terms of the possible role of PPIX-containing membraneous systems in primitive photosynthesis.     

Core-cross-linked polymeric micelles as paclitaxel carriers

Cross-linkable di- and triblock copolymers of poly(epsilon-caprolactone) (PCL) and monomethoxyl poly(ethylene glycol) (MPEG) were synthesized. These amphiphilic copolymers self-assembled into nanoscale micelles capable of encapsulating hydrophobic paclitaxel in their hydrophobic cores in aqueous solutions. To further enhance their thermodynamic stability, the micelles were cross-linked by radical polymerization of the double bonds introduced into the PCL blocks. Reaction conditions were found to significantly affect both the cross-linking efficiency and the micelle size. The encapsulation of paclitaxel into the micelles was confirmed by the proton nuclear magnetic resonance (1H NMR) spectroscopy. Encouragingly, paclitaxel-loading efficiency of micelles was enhanced significantly upon micelle core-cross-linking. Both the micelle size and the drug loading efficiency increased markedly with increasing the PCL block lengths, no matter if the micelles were core-cross-linked or not. However, paclitaxel-loading did not obviously affect the micelle size or size distribution. The cross-linked micelles exhibited a significantly enhanced thermodynamic stability against dilution with aqueous solvents. The efficient cellular uptake of paclitaxel loaded in the nanomicelles was demonstrated by confocal laser scanning microscopy (CLSM) imaging. This new biodegradable nanoscale carrier system merits further investigations for parenteral drug delivery.     

Reverse micelles in protein separation: the use of silica for the back-transfer process

In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein-loaded reverse micellar solution with a water phase containing an appropriate salt (back-transfer). In this article we describe an alternative "back extraction" procedure which is based on the addition of silica to the protein-containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60-80 of the total protein (alpha-chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80-100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short-chain lecithin was used. It is shown that the recovery of a alpha-chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity. (c) 1993 John Wiley & Sons, Inc.     

Reverse micelles of dipalmitoyl phosphatidyl choline in chloroform and their interactions with dapsone.

1H NMR studies of DiPalmitoyl Phosphatidyl Choline (DPPC) in CDCl3 at various concentrations indicate that DPPC exists as reverse micelles for concentrations beyond 6 mM. The chemical environments of the two acyl chains of DPPC are inequivalent and the inequivalence decreases with increasing DPPC concentration. At low concentrations of DPPC (less than 1.0 mM) intramolecular interactions predominate, whereas at high concentrations, intermolecular interactions predominate. Addition of water to this system, at high concentrations of the phospholipid, reduces the intermolecular interactions. In the presence of the antileprotic drug, Diamino Diphenyl Sulfone (DDS or Dapsone), significant shifts were observed only in the choline resonances of DPPC and the amino resonance of the drug, showing that the amino group of DDS interacts with the head group of DPPC.     

Reverse migration as a cause of vagrancy

Capsule Reverse migration in autumn does not occur to the same degree in all species of migrants, but is related to migratory direction.

Aims To identify factors determining degree of reverse migration and specifically to test whether it occurs in long-distance migrant species irrespective of their standard (normal) migration direction. Methods Multiple regression analysis on the number of individuals occurring as reverse migrants observed in northwest European countries.

Methods Multiple regression analysis on the number of individuals occurring as reverse migrants observed in northwest European countries.

Results The number of reverse migrants observed in northwest European countries is strongly correlated with standard migratory direction, estimated population size and detectability, but an effect of the distance travelled from the breeding areas is not supported. The pattern holds true for subsamples of the data set, including British and Irish records or Scandinavian records only, and when controlling for phylogeny.

Conclusion Birds that migrate eastward in autumn from their breeding grounds, mostly in eastern Europe, are more likely to consistently reverse migrate than those species migrating southward mostly from southern Europe.     

Reverse computation for rollback-based fault tolerance in large parallel systems

Reverse computation is presented here as an important future direction in addressing the challenge of fault tolerant execution on very large cluster platforms for parallel computing. As the scale of parallel jobs increases, traditional checkpointing approaches suffer scalability problems ranging from computational slowdowns to high congestion at the persistent stores for checkpoints. Reverse computation can overcome such problems and is also better suited for parallel computing on newer architectures with smaller, cheaper or energy-efficient memories and file systems. Initial evidence for the feasibility of reverse computation in large systems is presented with detailed performance data from a particle (ideal gas) simulation scaling to 65,536 processor cores and 950 accelerators (GPUs). Reverse computation is observed to deliver very large gains relative to checkpointing schemes when nodes rely on their host processors/memory to tolerate faults at their accelerators. A comparison between reverse computation and checkpointing with measurements such as cache miss ratios, TLB misses and memory usage indicates that reverse computation is hard to ignore as a future alternative to be pursued in emerging architectures.     

Conformational preferences of Leu-enkephalin in reverse micelles as membrane-mimicking environment

Enkephalin represents one of the bioactive peptide molecules most extensively investigated both in solution and in the crystal state. Depending upon the environment chosen for such studies, three main conformational states were identified for this flexible, linear pentapeptide—i.e., an extended conformation, a single-bend, and a double-bend structure. Since CD and Fourier transform ir (FTIR) spectra of Leu-enkephalin solubilized in negatively charged reverse micelles of bis (2-ethylhexyl)sulfosuccinate sodium salt/isooctane/water were supportive of a restricted conformational space of the aromatic side chains and of a bended type fold, we have analyzed by nmr the conformational preferences of Leu-enkephalin in reverse micelles using a synthetic [¹³C, ¹⁵N]-backbone-labeled sample. The overall conformation derived from nuclear Overhauser effect spectroscopy (NOESY) and ¹⁵N-filtered rotating frame NOESY (ROESY) spectra and by distance geometry calculations is a double-bend fold of the backbone that is comparable to one of the known x-ray structures. Thereby the tyrosine side chain is inserted into the hydrophobic core of the reverse micelles in a restrained conformational space as well evidenced by NOEs between the aromatic ring protons and the surfactant. The proximity of the aromatic rings of tyrosine and phenylalanine indicate a preferred structure consistent with the postulated conformation of the opioid peptide in the δ-receptor-bound state. These results confirm the interesting and promising properties of reverse micelles as membrane mimetica. © 1997 John Wiley & Sons, Inc. Biopoly 41: 591–606, 1997     

Relaxation phenomena in systems of protein-containing reverse micelles of surfactants in organic solvents

The research was aimed to establish the equilibrium processes in protein-containing systems of AOT reverse micelles in octane. As chromophore label for tracing the kinetics of the process, the acid-base indicator, p-nitrophenol, was used. The establishing of the equilibrium in the reverse micelle system notably decelerated in the presence of a solubilized protein (native and stearoylated alpha-chymotrypsin). During the establishing of the equilibrium, the solubilized enzyme can be irreversibly inactivated. The level of the residual activity of the enzyme in the equilibrium system depended on the procedure of micellar system preparation. The methods have been offered to set up the equilibrium in the reverse micelle system without inactivation of the solubilized enzyme.     

The nature of bile salt micelles as studied by deuterium NMR

Deuterium NMR of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid was studied. Molecular sizes obtained from deuterium spin-lattice relaxation time (T₁) data of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid in methanol and in water are in accordance with monomeric and tetrameric structures in the two media, respectively. The deuterium T₁ and intensity of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid in aqueous solution at pH 8.0–8.8 were studied as functions of NaCl and lecithin concentrations. The results indicated that tetramers are in equilibrium with larger aggregates when secondary micelles are formed in the precense of NaCl, and that 3α, 12α-dihydroxy-7,7-dideutero-5β-cholanoic acid forms mixed micelles with lecithin with a molecular ratio of 2 : 3.     

An immunoassay utilizing the DNA-coated polydiacetylene micelles as a signal generator

Immunoassay is an important technique to detect the disease biomarkers and pathogenic biological agents which often present at low levels in clinical samples. To improve sensitivity of the immunoassay, here we described the DNA-coated, nano-sized micelles in which the DNA strands play a role as signal generators in an immunoassay. This micelle-based immunoassay was evaluated for quantitation of a liver cancer biomarker and the sensitivity of the method was compared with those of the conventional methods.     

Polymeric micelles with water-insoluble drug as hydrophobic moiety for drug delivery

The hydrophobic block of polymeric micelles formed by amphiphilic copolymers has no direct therapeutical effect, and the metabolites of these hydrophobic segments might lead to some unexpected side effects. Here the hydrophobic core of polymeric micelles is replaced by highly water-insoluble drugs themselves, forming a new micellar drug delivery system. By grafting hydrophobic drugs of paclitaxel (PTX) onto the surface of hydrophilic hyperbranched poly(ether-ester) (HPEE), we constructed an amphiphilic copolymer (HPEE-PTX). HPEE-PTX could self-assemble into micellar nanoparticles in aqueous solution with tunable drug contents from 4.1 to 10.7%. Moreover, the hydrolysis of HPEE-PTX in serum resulted in the cumulative release of PTX. In vivo evaluation indicated that the dosage toleration of PTX in mice had been improved greatly and HPEE-PTX micellar nanoparticles could be used as an efficient prodrug with satisfactory therapeutical effect. We believe that most of the lipophilic drugs could improve their characters through this strategy.