Detection of Water in Lipase-Catalyzed Reactions in Reverse Micelles as Studied by Infrared Spectroscopy

The hydrolytic activity of lipolytic enzymes in reverse micelles can be measured continuously with Fourier Transform infrared spectroscopy (FTIR) by following in the region of the OH-stretching band the water consumption during the reaction. This possibility is unique to reverse micellar solutions, because they are optically transparent and because they contain only a limited amount of water.     

Decontamination of surfaces by lysozyme encapsulated in reverse micelles

Cells and enzymes can be used to decontaminate soil, water supplies, personal equipment, weapons and hospital equipment that have been exposed to bacteria, toxins or viruses. One of the problems associated with the use of microorganisms and enzymes for decontamination purposes is that the presence of water is not acceptable for some applications such as electronic equipment. One way of circumventing this problem is to allow the enzyme to distribute between a water phase and an organic phase-containing surfactant and then use the encapsulated enzyme in reverse micelles directly into the device to be clean. Reverse micelles were used to deliver the enzyme (lysozyme) to the cell-surface interface. They serve as a way to increase the local concentration of lysozyme and decrease the amount of water delivered. Specifically, we explored the lysis by free lysozyme and lysozyme encapsulated in reverse micelles of Klebsiella pneumoniae and Staphylococcus epidermidis attached to steel, glass, and hydroxyapatite. These two bacteria have been selected because they are known to be pathogenic and because of their differences in cell wall structure. Lysozyme was added to the surfaces in either reverse micelles or as a free solution and was tested under conditions of stirring and no stirring. Stirring was implemented to study the interplay between mass transfer limitations and surface roughness. We have shown that free lysozyme or lysozyme encapsulated in reverse micelles is capable of decontaminating surfaces of different texture. Lysis of the cells is slower when the encapsulated enzyme is used but lysis is more complete.     

The control of food flow in a society of the ant Myrmica rubra L

The control of prey, thin syrup and water flow, through a society of Myrmica was studied. Larval intake increases if they are deprived of prey, but not if they are deprived of water or sugar. Deprivation causes them to take prey juices from workers and they get more if the workers themselves have also been deprived; this is because such workers over-collect and readily pass on their surplus. Even well-fed larvae will take prey juices from these surfeited workers; they will also take sugary fluids but not water. The head of a larva elicits some food collection by workers even if it is immobile, but the real cause of food flow towards larvae is their ability to absorb and assimilate the prey juices which they can obtain from workers. Starved nurse workers can obtain prey and water from foragers but a reverse flow does not occur; only thin syrup is exchanged freely between workers.     

Chiral epoxide production using mycobacterium solubilized in a water-in-oil microemulsion

The application of many biotransformation processes is limited because the substrates/products are poorly water soluble, can be further metabolized, or are inhibitory. Hence non-aqueous media (e.g. two-phase systems, low water environments) are being examined to determine whether they can be used to overcome these problems. One novel approach is to encapsulate whole cells in water-in-oil (w/o) microemulsions (reverse micelles). In this study we have investigated the influence of key system parameters on system stability and epoxidation activity of Mycobacterium M156 cells in reverse micelles comprised of a mixture of Tween 85 and Span 80 (10-20 w%, with an hydrophilic/lipophilic balance [HLB] of 10 and a weight ratio of Tween 85 to Span 80 = 5.7) in n-hexadecane. It was found that the minimum allyl phenyl ether (APE) concentration required in the bulk hexadecane solvent phase for epoxidation to occur was 15 mM, whereas the minimum molar ratio of water to surfactant (W(0)) was 35. The optimum epoxidation rate achieved was 3.8 nmol/mg dwt-min with an APE concentration of 50 mM, and a W(0) of 50, with an enantiomeric excess (ee) of 86%. However, epoxidation was found to terminate approximately 3 h after initiation, and the causes for this were postulated to be either: the deleterious effect of the solvent on the Mycobacteria; inactivation of the energy generating system; an insufficient energy supply, or; the instability of the monooxygenase enzyme. It was concluded that on balance emulsion systems are not an economically viable system for producing phenyl glycidyl ether (PGE).     

Preparation of encapsulated proteins dissolved in low viscosity fluids

The majority of proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. One potential approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques is to encapsulate them in a reverse micelle which is dissolved in a low viscosity fluid. Unfortunately, promising low viscosity fluids such as the short chain alkanes, supercritical carbon dioxide, and various halocarbon refrigerants all require the application of significant pressure to be kept liquefied at room temperature. Here we describe the design and use of a simple cost effective NMR tube suitable for the preparation of solutions of proteins encapsulated in reverse micelles dissolved in such fluids.     

Reverse flow of sap in tree roots and downward siphoning of water by Grevillea robusta

1. Constant-power heat-balance sap flow gauges were used to compare sap flow in vertical and lateral roots of Grevillea robusta trees growing without access to ground water at a semiarid site in Kenya.
2. Reversal of sap flow occurred when root systems crossed gradients in soil water potential. Measurement of changes in the direction of flow was possible because of the symmetrical construction of the sap flow gauges; gradients in temperature across the gauges, and thus computed rates of sap flow, changed sign when reverse flow occurred.
3. Reverse flow in roots descending vertically from the base of the tree occurred, while uptake by lateral roots continued, when the top of the soil profile was wetter than the subsoil. The transfer of water downwards by root systems, from high to low soil water potential, was termed 'downward siphoning'; this is the reverse of hydraulic lift.
4. Downward siphoning was induced by the first rain at the end of the dry season and by irrigation of the soil surface during a dry period.
5. Downward siphoning may be an important component of the soil water balance where there are large gradients in water potential across root systems, from a wet soil surface downwards. By transferring water beyond the reach of shallow-rooted neighbours, downward siphoning may enhance the competitiveness of deep-rooted perennials.     

Purification and concentration of alkaline phosphatase by selective permeabilization of Escherichia coli using reverse micellar solutions

Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible.     

Sap Ascent in Vascular Plants: Challengers to the Cohesion Theory Ignore the Significance of Immature Xylem and the Recycling of Munch Water

In recent years the cohesion theory has been attacked on thegrounds that direct measurements made with the pressure probeindicate that sap tensions are much less (maximum tension approx.0.7 MPa) than indicated by parallel measurements made with themore conventional methods: osmotic methods, pressure bomb, orpsychrometer. It has also been claimed that other direct methodsdo not support the cohesion theory. Thus a re-examination usingthe Renner technique indicated sap tensions of approx. 2.5 MPa.Also an independent method based on mercury penetrometry providesevidence that sap tensions of at least 2.0 MPa can be demonstrateddirectly implying, that serious limitations arise from the pressureprobe method itself. Without tensions exceeding 2.0 MPa mangroveswould be unable to extract fresh water for transpiration fromseawater. It is suggested that the pressure probe is susceptibleto bias because it investigates the least mature xylem conduitswhile they are still under varying degrees of turgor pressureand only partially interconnected with the main xylem system.This supposition is supported by claims that the xylem sap sampledby the probe contains significant concentrations of solutes.Additionally water, supplied by reverse osmosis from the sievetubes (‘Münch water’), is continually beingliberated in the vicinity of the outermost xylem vessels hydratingthem to an atypical degree which can explain several of thediscrepancies claimed. These results, which are supported bythe work of others, demonstrate that the challenges to the cohesiontheory for the ascent of sap are ill-founded. The release ofwater from the phloem can explain not only some discrepanciesclaimed by the cohesion challengers, but also explain the refillingof cavitated xylem conduits: a hitherto unsuspected role forthe phloem transport system. Cohesion theory; sap ascent; cavitation; pressure probe; xylem transport; vessel development; recycled water; reverse osmosis     

Kinetic behaviour of alpha-chymotrypsin in reverse micelles. A stopped-flow study.

At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of alpha-chymotrypsin have been studied in reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. Pre-steady state and steady-state kinetic constants, in water and in reverse micelles, have been determined by stopped-flow spectrophotometry for the hydrolysis of two substrates, namely acetyl-L-tryptophan-p-nitrophenyl ester and p-nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k2) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kcat) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.     

Enzymes in Low Water Systems

Water is fundamental for enzyme action and for formation of the three-dimensional structure of proteins. Hence, it may be assumed that studies on the interplay between water and enzymes can yield insight into enzyme function and formation. This has proven correct, because the numerous studies that have been made on the behavior of water-soluble and membrane enzymes in systems with a low water content (reverse micelles or enzymes suspended in nonpolar organic solvents) have revealed properties of enzymes that are not easily appreciated in aqueous solutions. In the low water systems, it has been possible to probe the relation between solvent and enzyme kinetics, as well as some of the factors that affect enzyme thermostability and catalysis. Furthermore, the studies show that low water environments can be used to stabilize conformers that exhibit unsuspected catalytic properties, as well as intermediates of enzyme function and formation that in aqueous media have relatively short life-times. The structure of enzymes in these unnatural conditions is actively being explored.     

Tryptophan fluorescence studies of melanotropins in the amphiphile-water interface of reversed micelles

We report studies on the interaction of α-melanocyte stimulating hormone (α-MSH) and a synthetic analogue (MSH-I) with reverse micelles prepared from the amphiphilic sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. The tripeptide lysyl-tryptophyl- lysine and the isolated amino acid tryptophan were also investigated as simpler compounds interacting with the micelles. Tryptophan fluorescence parameters (spectral position of emission band, anisotropy, and lifetime decay) demonstrated that in the presence of reverse micelles the environment around the fluorophore is less polar and more rigid than bulk water. Those parameters are sensitive to the changes induced in the micelles by the presence of water. In large micelles having a water/amphiphile molar ratio above 10, the modifications detected by fluorescence are small and the location of the fluorophore is not affected by a further increase in the concentration of the bulk water. The results, with additional support from quenching experiments, indicated that the different compounds occupy different positions in the large reverse micelles, but in any case they are in the interface region, without dispersing into the bulk water. From decay associated spectra, conformations were identified showing different degrees of tryptophan exposition to polar and nonpolar local environments. The conformation related to the long lifetime has its tryptophan more exposed to water while that associated to the intermediate lifetime has that residue stabilized in nonpolar media. The native hormone α-MSH and the analogue MSH-I present similar conformations in dry micelles. However, in buffer and in the large hydrated micelles, differences in conformations are evident, and could be related to the different physiological activity of the peptides. Received: 4 August 1999 / Revised version: 17 December 1999 / Accepted: 4 January 2000     

植物水通道的生理生态特性及其参与气孔运动的研究进展

植物水通道对水分运输具有专一性, 能够调节细胞中水分、一些离子和其他小溶质的转运, 因而在植物的生长发育中发挥着重要作用。本文综述了植物水通道的研究进展, 重点介绍了植物水通道的分子特性和生理生态特性及其在植物气孔运动中的作用, 讨论了水通道在气孔振荡中的作用和地位。     

Kinetic model for enzymatic hydrolysis in reverse micelles

A bound water model is developed for the interpretation of kinetic data of b-galactosidase in reverse micelles. Assessing the kinetic parameters of p-nitrophenyl-b-D-galactopyranoside hydrolysis in aqueous and reverse micellar system reveals that the major effect on hydrolytic rate is owing to the amount of free water in reverse micelles, not the enzyme molecules' structural change.     

植物水通道的生理生态特性及其参与气孔运动的研究进展

植物水通道对水分运输具有专一性,能够调节细胞中水分、一些离子和其他小溶质的转运,因而在植物的生长发育中发挥着重要作用.本文综述了植物水通道的研究进展,重点介绍了植物水通道的分子特性和生理生态特性及其在植物气孔运动中的作用,讨论了水通道在气孔振荡中的作用和地位.     

Relative Humidity and the Killing of Bacteria: The Survival of Damp Serratia marcescens in Air

The viability of washed moist cells of Serratia marcescens after storage has been measured in relation to variations in the prior treatment of the cells and in conditions of storage. The factors considered were: (i) water content during storage; (ii) method of arriving at water content (partial drying in vacuum or freeze-drying and addition of water); (iii) presence or absence of air during storage.

Increasingly rapid decay occurs as the water content at which the cells are stored is diminished from above 90% to 20 or 30% (“critical” water content). It occurs in presence or absence of air and it occurs whether the final water content is approached by removal of water from wet cells or by addition of water to freeze-dried cells.

The rate of decay during storage at 20 to 30% water is somewhat diminished by the presence of air (“protective” effect of air).

As the water content is further reduced to less than 10%, the stability of cells stored in a vacuum approaches that of wet cells. In presence of air the reverse is true: the stability decreases until at less than 1% water, the decay rate is about as great as at the “critical” water content (“toxic” effect of air).

Particularly rapid decay of S. marcescens at the “critical” water content has escaped attention in aerosol studies because accurate control of relative humidity (RH) in this region, RH 94 to 99%, is virtually impossible in such studies. On the other hand, values of decay rates referred to measured water contents are quite unreliable in the 20 to 80% RH zone because the corresponding variation of water content is too small to measure reliably. Thus data of the kind reported in this paper cannot be directly compared to the published results of studies of air-borne bacteria, although they are relevant to the practical question of air-borne infection in humid atmospheres.

     

气生植物的生物学特性及研究展望

气生植物是指不需要土壤,生长所需的水分和营养可以全部来自空气的植物。它既不同于附生植物．也不同于具有气生根的植物。主要包括地衣、苔藓、蕨、凤梨科和兰科植物中的某些附生类群。它们没有根或者根不发达,仅起固定植株的作用。因为气生植物直接从空气中吸收水分和养分,但空气中的水分和养分毕竞是有限的,所以这些植物一般都具有很强的利用水分及养分的能力,很多植物已经成为有效地检测环境变化的“指示生物”和去除环境污染的修复植物。另外,因为这些植物具有忍受恶劣环境条件的生理基础,还可能成为适应空间环境的先锋植物,在空间植物学研究中将具有特殊的意义。     

The mechanism of restructuring of surfactant monolayer on mica surface in aqueous solution: molecular dynamics simulation

Molecular dynamics simulation was employed to investigate the restructuring process of CTAB monolayer at mica/water interface. The reversing process of CTAB monolayer was exploited by diffusion of water molecules, reversing of CTAB molecules with time evolution and restructuring of the surfactant monolayer. The results showed that bromide ions around surfactant head groups diffused into bulk water readily due to the electrostatic repulsion caused by negatively charged mica surface. Meanwhile, because of the electrostatic attraction between water molecules and mica surface, part of water molecules can penetrate the surfactant monolayer to form water channel which bridges bulk water and mica surface. The monolayer structure was disturbed by diffusion of bromide ions and formation of water channel. Few of the head groups of surfactants tended to reverse and enter into aqueous solution. The number of reversed surfactant molecules increased with time evolution. The monolayer restructured into bilayer structure gradually. Finally, a cylindrical aggregate was obtained.     

Solubilization of soybean mitochondria in AOT/isooctane water-in-oil microemulsions

The water-in-oil microemulsion system bis(2 ethyl-hexyl-sodium-succinate (AOT)/isooctane/water is able to solubilize soybean nodules mitrochrondria. Transparent and thermodynamically stable hydrocarbon solutions are obtained, which can be assayed for mitochondrial activity just as aqueous solutions. Malate dehydrogenase (MDH) activity was measured in vivo and gave in reverse micelles very similar results as in water. However the kinetic behavior of this reaction in AOT/isooctane reverse micelles shows some differences with respect to water. Mitochondria in reverse AOT micelles are able to retain about 70% of their initial MDH activity after three days. Mitochondria can be back-transferred from reverse micelles to water and show respiratory activity almost identical to the native organelles. Electron microscopy studies show that the dimensions of mitochondria back-transferred into water from AOT micelles are comparable to the dimensions of the native organelles.