Low Water Environments Research Articles

Overexpression in a non-native halophilic host and biotechnological potential of NAD+-dependent glutamate dehydrogenase from Halobacterium salinarum strain NRC-36014

Enzymes produced by halophilic archaea are generally heat resistant and organic solvent tolerant, and accordingly important for biocatalytic applications in 'green chemistry', frequently requiring a low-water environment. NAD(+)-dependent glutamate dehydrogenase from an extremely halophilic archaeon Halobacterium salinarum strain NRC-36014 was selected to explore the biotechnological potential of this enzyme and genetically engineered derivatives. Over-expression in a halophilic host Haloferax volcanii provided a soluble, active recombinant enzyme, not achievable in mesophilic Escherichia coli, and an efficient purification procedure was developed. pH and salt dependence, thermostability, organic solvent stability and kinetic parameters were explored. The enzyme is active up to 90°C and fully stable up to 70°C. It shows good tolerance of various miscible organic solvents. High concentrations of salt may be substituted with 30% DMSO or betaine with good stability and activity. The robustness of this enzyme under a wide range of conditions offers a promising scaffold for protein engineering.

Long-term acclimation of mesophyll conductance, carbon isotope discrimination and growth in two contrasting Picea asperata populations exposed to drought and enhanced UV-B radiation for three years

Two Picea asperata Mast. populations originating from wet and dry climate regions of China were subjected to enhanced UV-B radiation, drought and their combination in a greenhouse for 3 years. Drought treatment caused a significant decrease in photosynthesis, mesophyll conductance ( g m), carbon isotope discrimination ( Δ) and growth characteristics when compared with well-watered treatment. In contrast to the great effects of drought stress, enhanced UV-B radiation showed some but not as substantial negative effects on these parameters. Little interaction effect between drought and UV-B radiation was detected, and the drought effect in combination with enhanced UV-B was not more pronounced than with no enhanced UV-B radiation. These results suggest that enhanced UV-B radiation does not aggravate the drought effect on P. asperata seedlings. The results also showed that the proportional role of the intercellular CO 2 ( C i) decreased, while the role of chloroplastic concentration ( C c) became increasingly important in explaining the high values of carbon isotope composition ( δ 13C), when the water stress progressed in time. In addition, multivariate causal models proposed that there is a direct causal relationship between specific leaf area (SLA) and g m, which is not mediated by leaf N. Besides, there are functional links between g m and photosynthetic capacity ( V cmax). On the other hand, the study showed that net assimilation rate (NAR) is the main driving force for changes in relative growth rate (RGR), especially in low-water environments, and the degree of acclimation of respiration in the light ( R L) is of central importance to the greater role played by NAR in determining variation in RGR.

The influence of cation, anion and water content on the rate of formation and pore size distribution of zeolite ZSM-5

Rapid synthesis and high mesoporosity of ZSM-5 is promoted by altering a fixed molar regime with respect to anion and cation type and water content only. This study shows that merely changing the cation and anion types, accompanying the hydroxide and alumina (Al) sources, respectively, has an impact on the characteristics of ZSM-5. Under identical and replicated preparation and testing conditions, the synthesis time, Al incorporation, product yield, acidity, morphology, pore size distribution and catalytic activity were affected. An ion pair combination of Na + /NO3 - allowed the formation of ZSM-5 with enhanced mesoporosity withi n3hi n a high-water environment, whereas ZSM-5 prepared with the Na + /SO4 2- combination had enhanced n-hexane cracking activity. Use of a K + /NO3 - ion pair combination in a high-water environment resulted in slower time of formation and larger ZSM-5 crystals with extra-framework Al, loss of acidity, lower surface areas and reduced n-hexane cracking activity. The sulphate anion inhibited the incor- poration of Al in ZSM-5 products in low-water environments. Low- water levels increased the time of formation of ZSM-5. : zeolite ZMS-5, synthesis, cation, anion, acidity, poros- ity, extra-framework aluminium, n-hexane cracking activity, sul- phate, nitrate

Phenotypic plasticity and performance of Taraxacum officinale (dandelion) in habitats of contrasting environmental heterogeneity

Ecological theory predicts a positive association between environmental heterogeneity of a given habitat and the magnitude of phenotypic plasticity exhibited by resident plant populations. Taraxacum officinale (dandelion) is a perennial herb from Europe that has spread worldwide and can be found growing in a wide variety of habitats. We tested whether T. officinale plants from a heterogeneous environment in terms of water availability show greater phenotypic plasticity and better performance in response to experimental water shortage than plants from a less variable environment. This was tested at both low and moderate temperatures in plants from two sites (Corvallis, Oregon, USA, and El Blanco, Balmaceda, Chile) that differ in their pattern of monthly variation in rainfall during the growth season. We compared chlorophyll fluorescence (photosynthetic performance), flowering time, seed output, and total biomass. Plants subjected to drought showed delayed flowering and lower photosynthetic performance. Plants from USA, where rainfall variation during the growth season was greater, exhibited greater plasticity to water shortage in photosynthetic performance and flowering time than plants from Chile. This was true at both low and moderate temperatures, which were similar to early- and late-season conditions, respectively. However, phenotypic plasticity to decreased water availability was seemingly maladaptive because under both experimental temperatures USA plants consistently performed worse than Chile plants in the low water environment, showing lower total biomass and fewer seeds per flower head. We discuss the reliability of environmental clues for plasticity to be adaptive. Further research in the study species should include other plant traits involved in functional responses to drought or potentially associated with invasiveness.

Environmental Heterogeneity and Population Differentiation in Plasticity to Drought in Convolvulus Chilensis (Convolvulaceae)

Plant populations may show differentiation in phenotypic plasticity, and theory predicts that greater levels of environmental heterogeneity should select for higher magnitudes of phenotypic plasticity. We evaluated phenotypic responses to reduced soil moisture in plants of Convolvulus chilensis grown in a greenhouse from seeds collected in three natural populations that differ in environmental heterogeneity (precipitation regime). Among several morphological and ecophysiological traits evaluated, only four traits showed differentiation among populations in plasticity to soil moisture: leaf area, leaf shape, leaf area ratio (LAR), and foliar trichome density. In all of these traits plasticity to drought was greatest in plants from the population with the highest interannual variation in precipitation. We further tested the adaptive nature of these plastic responses by evaluating the relationship between phenotypic traits and total biomass, as a proxy for plant fitness, in the low water environment. Foliar trichome density appears to be the only trait that shows adaptive patterns of plasticity to drought. Plants from populations showing plasticity had higher trichome density when growing in soils with reduced moisture, and foliar trichome density was positively associated with total biomass.

Glass-fiber disks provide suitable medium to study polyol production and gene expression in Eurotium rubrum

Eurotium species often dominate the fungal population in stored grain and are responsible for spoilage. In this study we tested the usefulness of glass fiber disks to aid the analysis of growth, polyol content and gene expression in E. rubrum in response to various water activities. Growth measurements based on ergosterol content and conidial production indicated that E. rubrum grew as well at 0.86 aw as 0.98 aw. The rate of growth was considerably reduced at 0.83 aw and 0.78 aw. In contrast, under our conditions, Aspergillus flavus and A. nidulans were able to grow only in the highest water activity (0.98 aw). Mannitol was the predominant polyol in all three fungal species grown at 0.98 aw. When E. rubrum was grown at 0.86 aw or lower, glycerol comprised greater than 90% of the total polyols. After a shift from 0.86 aw to 0.98 aw, mannitol levels in E. rubrum increased to 89% of the total polyols within 24 h. Of six genes whose expression was measured by quantitative real-time PCR, three were affected by water activity. Expression of putative hydrophobin and mannitol dehydrogenase genes was higher at 0.98 aw than at 0.86 aw. A putative triacylglycerol lipase gene was expressed at higher levels in 0.86 aw. The results of this study indicate that the disk method is suitable to study the effects of water activity on growth, polyol biosynthesis and gene expression in E. rubrum. The results also indicate the potential competitiveness of E. rubrum over A. flavus and A. nidulans in low water environments associated with stored grain.

Glass-fiber disks provide suitable medium to study polyol production and gene expression in Eurotium rubrum

Eurotium species often dominate the fungal population in stored grain and are responsible for spoilage. In this study we tested the usefulness of glass fiber disks to aid the analysis of growth, polyol content and gene expression in E. rubrum in response to various water activities. Growth measurements based on ergosterol content and conidial production indicated that E. rubrum grew as well at 0.86 aw as 0.98 aw. The rate of growth was considerably reduced at 0.83 aw and 0.78 aw. In contrast, under our conditions, Aspergillus flavus and A. nidulans were able to grow only in the highest water activity (0.98 aw). Mannitol was the predominant polyol in all three fungal species grown at 0.98 aw. When E. rubrum was grown at 0.86 aw or lower, glycerol comprised greater than 90% of the total polyols. After a shift from 0.86 aw to 0.98 aw, mannitol levels in E. rubrum increased to 89% of the total polyols within 24 h. Of six genes whose expression was measured by quantitative real-time PCR, three were affected by water activity. Expression of putative hydrophobin and mannitol dehydrogenase genes was higher at 0.98 aw than at 0.86 aw. A putative triacylglycerol lipase gene was expressed at higher levels in 0.86 aw.. The results of this study indicate that the disk method is suitable to study the effects of water activity on growth, polyol biosynthesis and gene expression in E. rubrum. The results also indicate the potential competitiveness of E. rubrum over A. flavus and A. nidulans in low water environments associated with stored grain.

Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water.

We have examined enzymes in nearly anhydrous organic solvents spanning a wide range of dielectric constants using a combination of electron paramagnetic resonance (EPR) spectroscopy, molecular dynamics simulations, high-pressure kinetic studies and the electrostatic model of Kirkwood. This approach enabled us to investigate the relationship between catalytic activity, protein flexibility and solvent polarity for an enzymatic reaction proceeding through a highly polar transition state in the near absence of water. Further insights into water-protein interactions and the involvement of water in enzyme structure and function have been obtained by EPR and multinuclear nuclear magnetic resonance studies of enzymes suspended and immobilized in organic solvents with and without added water. In these systems, correlations were observed between the water content and enzyme activity, flexibility, and active-site polarity, although the structural properties of suspended and immobilized enzymes differed markedly. These results have helped to elucidate the role of water in molecular events at the enzymic active site leading to improved biocatalysis in low-water environments.

Optimization of Hydrolase Efficiency in Organic Solvents.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Optimization of hydrolase efficiency in organic solvents.

The application of hydrolases in organic solvents for synthetic purposes is a procedure routinely adopted in organic chemistry, especially for the preparation of chiral building blocks. Numerous studies have shed light on several aspects of the mechanism of hydrolase action in low-water environments. Procedures suitable to improve the catalytic efficiency of enzymes and productivity of the synthetic processes have been reported. These fundamental and applied investigations have made hydrolase-catalyzed reactions in organic solvents of industrial interest. In this article we describe and discuss various approaches adopted to optimize the performance of hydrolases in organic media, with special emphasis on the formulation of the biocatalysts which, under proper conditions, can display an activity equal to that displayed in aqueous buffers.

Mechanism of adaptation of an atypical alkaline p-nitrophenyl phosphatase from the archaeon Halobacterium salinarum at low-water environments.

Enzymes suspended in organic solvents represent a versatile system for studying the involvement of water in catalytic properties and their flexibility in adapting to different environmental conditions. The extremely halophilic alkaline p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum was solubilized in an organic medium consisting of reversed micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as cosurfactant. Hydrolysis of p-nitrophenylphosphate was nonlinear with time when the enzyme was microinjected into reversed micelles that contained substrate. These data are consistent with a kinetic model in which the enzyme is irreversibly converted from an initial form to a final stable form during the first seconds of the encapsulation process. The model features a rate constant (k) for that transition and separate hydrolysis rates, v(1) and v(2), for the two forms of the enzyme. The enzyme conversion may be governed by the encapsulation process.

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).

Effect of beta-galactosidase hydration on alcoholysis reaction in organic one-phase liquid systems.

Alcoholysis reactions were performed in organic one-phase liquid systems with E. coli beta-galactosidase to produce heptyl-beta-galactoside from lactose and 1-heptanol. The reaction rate was highly dependent on the amount of water solubilized in the alcohol. A larger amount of water led to a system of two liquid phases in which the alcoholysis rate was 73% faster than in the one-phase system. No hydrolysis reaction of either lactose or product was observed in one-phase liquid systems up to 20 h, independent of the water content. Solubility of lactose in the organic phase increased with the water content in the system and the reaction followed the Michaelis-Menten model. Water activity was calculated for heptanol containing different amounts of water and the obtained values were used to estimate the hydration of beta-galactosidase from known models. Enzyme activity correlated with sorbed water, similar to the behavior reported for lysozyme in low water environments. It is concluded that water contribution to enzyme hydration dominates the rate of reaction compared to its effect on lactose solubilization.

Effects of environmental stress on leaf hair density and consequences for selection

Abstract We explored how five different environmental stresses and a benign environment affect the phenotypic expression of leaf hairs, and the potential for evolutionary response in this trait. To address these questions we planted full-sib families that had been selected for stress tolerance in a factorial design in which selection history was fully crossed with the current environment (eight families × six selection histories × six current environments × three blocks=864 plants). Our data suggest that leaf hair density is a complex character composed of two separable traits: leaf area and the number of hairs initiated per leaf. Leaf size is primarily controlled by the growth environment, whereas leaf hair initiation shows more genetically based variation. In the control and low water environments there was a negative linear relationship between leaf size and leaf hair density. However, within the low light and high boron treatments, leaf hair density remained constant across a range of leaf sizes, suggesting that these stresses disrupt normal leaf hair development. Selection gradient analysis showed that leaf hair density and leaf size were significantly positively associated with fitness in 4/6 of our environments. Our results suggest that environmental variation may diminish the ability of herbivores and pathogens to cause directional selection on leaf hair density.

Evolutionary Divergences in Leaf Structure and Chemistry, Comparing Rainfall and Soil Nutrient Gradients

Leaf structural and chemical characteristics were compared in pairs of species that were phylogenetically independently contrasted along rainfall gradients (10 pairs) or soil nutrient gradients (9 pairs), using perennial plants in New South Wales, Australia. The objective was to test hypotheses regarding leaf attributes that should be successful in sustaining populations in vegetation at lower vs. higher rainfall or lower vs. higher soil nutrients, and especially to assess the proposition that lower rainfall and lower soil nutrients favor similar shifts in leaf attributes, and in this sense can be grouped together as sources of “stress.” Some evolutionary divergences in leaf structure occurred repeatedly toward the lower end of both rainfall and soil nutrient gradients. These include narrower leaves, lower specific leaf area (SLA), thicker lamina, and denser leaf tissue. In other respects, the different resource gradients appeared to favor different leaf attributes. In 8 of 10 contrasts, plants of low-water environments had more sclerified vasculature than their relatives on higher rainfall sites. This may be an adaptation to resist wilting and so mitigate cell damage. In 9 of 10 contrasts, plants of low-water environments had more nitrogen per unit leaf area than their relatives in high-rainfall sites. This may be to exploit higher light availability in arid communities. In 7 of 9 contrasts, plants from low soil nutrient environments had thicker epidermis than their relatives on higher nutrient soils. In 8 of 9 soil nutrient contrasts, leaf nitrogen concentration was lower in the low-nutrient species, and consequently, the ratios of phenol/N and tannin/N were higher. Thicker epidermis and increased defense/N ratios may reduce herbivory and thereby reduce demand on soil nutrients required to replace leaves.

EVOLUTIONARY DIVERGENCES IN LEAF STRUCTURE AND CHEMISTRY, COMPARING RAINFALL AND SOIL NUTRIENT GRADIENTS

Leaf structural and chemical characteristics were compared in pairs of species that were phylogenetically independently contrasted along rainfall gradients (10 pairs) or soil nutrient gradients (9 pairs), using perennial plants in New South Wales, Australia. The objective was to test hypotheses regarding leaf attributes that should be successful in sustaining populations in vegetation at lower vs. higher rainfall or lower vs. higher soil nutrients, and especially to assess the proposition that lower rainfall and lower soil nutrients favor similar shifts in leaf attributes, and in this sense can be grouped together as sources of “stress.” Some evolutionary divergences in leaf structure occurred repeatedly toward the lower end of both rainfall and soil nutrient gradients. These include narrower leaves, lower specific leaf area (SLA), thicker lamina, and denser leaf tissue. In other respects, the different resource gradients appeared to favor different leaf attributes. In 8 of 10 contrasts, plants of low-water environments had more sclerified vasculature than their relatives on higher rainfall sites. This may be an adaptation to resist wilting and so mitigate cell damage. In 9 of 10 contrasts, plants of low-water environments had more nitrogen per unit leaf area than their relatives in high-rainfall sites. This may be to exploit higher light availability in arid communities. In 7 of 9 contrasts, plants from low soil nutrient environments had thicker epidermis than their relatives on higher nutrient soils. In 8 of 9 soil nutrient contrasts, leaf nitrogen concentration was lower in the low-nutrient species, and consequently, the ratios of phenol/N and tannin/N were higher. Thicker epidermis and increased defense/N ratios may reduce herbivory and thereby reduce demand on soil nutrients required to replace leaves.

Calcium dependence of Pi phosphorylation of sarcoplasmic reticulum Ca 2+-ATPase at low water content: water dependence of the E 2→E 1 conversion

Enzymes entrapped in reverse micelles can be studied in low-water environments that have the potential of restricting conformational mobility in specific steps of the reaction cycle. Sarcoplasmic reticulum Ca 2+-ATPase was incorporated into a reverse-micelle system (TPT) composed of toluene, phospholipids, Triton X-100 and varying amounts of water (0.5–7%, v/v). Phosphorylation of the Ca 2+-ATPase by ATP required the presence of both water and Ca 2+ in the micelles. No phosphoenzyme (EP) was detected in the presence of EGTA. Phosphorylation by Pi (inorganic phosphate) in the absence of Ca 2+ was observed at water content below that necessary for phosphorylation by ATP. In contrast to what is observed in a totally aqueous medium, EP formed by Pi was partially resistant to dephosphorylation by Ca 2+. However, the addition of non-radioactive Pi to the EP already formed caused a rapid decrease in radiolabelled enzymes, as expected for the isotopic dilution, indicating the existence of an equilibrium (E+Pi↔EP). Phosphorylation by Pi also occurred in TPT containing millimolar Ca 2+ concentrations in a range of water concentrations (2–5% v/v). The substrates p-nitrophenyl phosphate, acetyl phosphate, ATP and GTP increased the EP level under these conditions. These results suggest that: (1) the rate of conversion of the ATPase conformer E 2 into E 1 is greatly reduced at low water content, so that E 2→E 1 becomes the rate-limiting step of the catalytic cycle; and (2) in media of low water content, Pi can phosphorylate both E 1Ca and E 2. Thus, the effect of enzyme hydration is complex and involves changes in the phosphorylation reaction at the catalytic site, in the equilibrium between E 2 and E 1 conformers, and in their specificity for substrates.

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.

Le Miocène du bassin de Saint-Florent(Corse): Modalités de la transgression du Burdigalien supérieur et mise en évidence du Serravallien

The Miocene series of Corsica deposited after the anti clockwise rotation of the Corsica-Sardinia block,between 18 and 21 Ma (Réhault 1981; Réhault et al. 1984). The study of the Saint-Florent basin (NW corsica) allows to document the conditions of the Miocene transgression and the evolution of the basin. The Saint-Florent sedimentary serie consist of 3 marine formations lying between 2 continental ones. At the base, the continental formation of Fium Albino (150 m) in the northern part of the basin, stratigraphically lies on the Upper Nebbio alpine nappe. It consists of well stratified, red, colluvial deposits from local origin, with interbedded palaeosoils remains. We have very few paleontological evidences, however from geometrical evidence, these series are clearly post nappe and ante Upper Burdigalian. This formation is described here for the first time. At the upper part, the continental formation of Saint-Florent, characterized by well rounded pebbles of rhyolites, lies with an erosional surface truncating different terms of the marine series. It is probably of Lower Tortonian age. The 3 lithostratgraphic units of the marine series are, from the base to the top: the Torra formation (50 m) is characterized by coastal deposits (conglomerate, siltstone, sandstone layers, with debris of oyster, sands with scutella) with sparse micro-fauna and - flora, suggesting an Upper Burdigalian (upper N7/lower N8 Blow's zones); the Monte San Angelo Formation (250 m) is a white calcarenite with rhodophytes, its different components - fine and coarse terrigenous detritus, benthic or planktonic bioclasts-having built hydraulic dunes under the action of varying currents in a low water environment. Planktonic foraminifera - rich levels indicate a Langhian age (from N8 to the upper part of the N9 Blow's zones); the Farinole Formation (100 m), described here for the first time, is observed only in the northern part of the basin. It consists of marls and clayey sandstones deposited in circalittoral environment. Rich microfauna of benthic and planktonic foraminifera, and rich calcareous nannoplankton evidence the Serravallian (NN7-NN6, Martini's zones). To the east, the basin is limited by a meridian normal fault. On the one hand, synsedimentary deformations in the Torra formation and in the lower part of the Monte San Angelo Formation are located to the north of the basin near the fault. On the other hand, thick subtidal environment deposits of the Torra Formation occure in a shallow water. Thus the subsidence of this Formation is probably controled by the syn-sedimentary activity of the meridian normal fault. Extensional fractures indicating a NE-SW direction of extension.

Reverse Micelle Systems Composed of Water, Triton X-100, and Phospholipids in Organic Solvents: 2. Catalysis and Thermostability of Hexokinase

Catalysis, stability, and thermostability of yeast hexokinase were determined in the microenvironments of two organic solvent/Triton X-100/phospholipids systems. In the abscence of enzyme, phase diagrams showed two transparent/turbid transitions, and reverse micelles were only observed in the second region of transparency (T2), where particle size as a function of water content shows a minima (see previous paper in this issue). In the present work, enzyme activity was detected throughout the four regions of the phase diagrams of these systems. Catalysis increased with water content; nevertheless, the maximum activities that were reached in the toluene and propylbenzene systems were 30 and 1.6%, respectively, of the activity in all aqueous media. Because in the T2 region in the propylbenzene system, micelles are much smaller than in toluene (see preceding paper), it would appear that expression of catalysis depends on the size of the micelles. However, a comparison of the dimensions of hexokinase and those of reverse micelles in the T2 region, suggests that in this region, hexokinase entrapment increases the inner volume of the micelle. High enzyme thermostability was only observed in the first transparent region (T1) of the system that contained phospholipids. In this region, hexokinase induced the formation of reverse micelles from dispersed surfactant monomers. There is a striking similarity in the dimensions of hexokinase entrapped in reverse micelles as determined by dynamic light scattering measurements in the T1 region with those of hexokinase as obtained from X ray diffraction studies of the enzyme in a crystalline environment. This suggest that high thermostability, and low catalytic rates result from restrictions in mobility imposed by a low water environment.