Malic dehydrogenase from tamarix roots: effects of sodium chloride in vivo and in vitro

Soluble and mitochondrial malic dehydrogenases (MDH) were isolated from root tips of the halophyte Tamarix tetragyna L. grown in the presence and absence of NaCl. The activity of the enzymes isolated from root tips grown in the presence of NaCl was lower than that of the enzymes isolated from roots grown in absence of NaCl. The mitochondrial MDH was much more sensitive to salinity than the soluble MDH. The soluble enzyme from roots grown in NaCl had a higher Km for malate and lower Km for NAD than enzyme from the control roots. Addition of NaCl in vitro at 72 mM significantly stimulated the reductive activity of soluble MDH, while higher NaCl concentrations (240 mM and above) depressed enzyme activity. The inhibition of enzyme activity by various salts was found to be in the order MgCl₂ > NaCl = KCl > Na₂SO₄. Mannitol at equiosmotic concentrations had no effect. Substrate inhibition, typical for oxaloacetate oxidation, was not observed at high NaCl concentrations in vitro and high substrate concentrations neutralized the inhibitory effect of NaCl. Increased coenzyme concentrations had no effect. In vitro NaCl increased the Km for malate and oxaloacetate already at relatively low concentrations. At the same time NaCl decreased the Km for NAD and NADH. The inhibitory effect of NaCl on enzyme activity seems not to be due to the effect on the Km alone. Soluble and mitochondrial MDH had different responses to pH changes, mitochondrial MDH being more sensitive. Mitochondrial MDH released from the particles had a similar response to that of the entire particles. Changes of pH modified the effect of NaCl on enzyme activity. It was postulated that NaCl apparently induces conformational changes in the enzyme.     

Characterization and distribution of a phospholipase A2 activity from adult rabbit lung

A phospholipase A₂ activity was characterized in adult rabbit lung. This activity was calcium- and deoxycholate-dependent and displayed an alkaline pH optimum. K_m and V_max were 0.176mM and 256.8 pmoles/min./mg protein respectively. The microsomal fraction displayed the highest enzymatic specific activity; the lowest activity was present in the cytosol. Yet this latter fraction accounted for the majority of the total activity. Although the specific activity was high within the lamellar body fraction this compartment contained only 2% of the total activity. Phospholipase A₂ activity was inhibited by bromophenacyl bromide, chlorpromazine and mepacrine in decreasing order of effectiveness. Treatment of the microsomes with increasing concentrations of NaCl indicated that the lung phospholipase A₂ activity was related loosely bound to the microsomal membranes and was maximally removed with salt at a concentration only slightly higher than physiological. Addition of calmodulin to the enzyme assay did not significantly alter hydrolysis of labelled phosphatidylcholine.     

Correlation between catalysis and tertiary structure arrangement in an archaeal halophilic subtilase

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M (NaCl), Nep behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3–1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.     

Salt Stress-induced Responses in Growth and Metabolism in Callus Cultures and Differentiating In Vitro Shoots of Indian Ginseng (Withania somnifera Dunal)

In vitro-grown shoots and calli of Withania somnifera, an important medicinal plant, were exposed to various types of salts under in vitro culture conditions. Membrane permeability, lipid peroxidation, and the antioxidant system increased in shoots as well as in unorganized callus tissues under all the three concentrations of KCl, NaCl, KNO₃, NaNO₃, and CaCl₂. The growth responses of shoots and callus cultures under various salt treatments revealed that the tissue could grow better under NaCl and KNO₃ compared to other salts and the in vitro shoots appeared healthy at 50?mM concentration of NaCl and KNO_3. The activity of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, lipoxygenase, polyphenol oxidase, and glutathione reductase increased under salt treatments, especially at higher concentrations. The greatest activity increase was recorded for peroxidases, whereas CAT was the least responsive. Only two isoforms, Mn-superoxide dismutase (Mn-SOD) and Fe-SOD, could be visualized in callus tissue while Cu/Zn-SOD was absent. Diaphorase 4 was totally missing in callus tissue and was detected only in shoots. Phenolics accumulated at all the concentrations of the salts tested as an induced protective response. The higher concentration of CaCl₂ produced maximum increases in antioxidants and enzymatic activities compared to other salts. Thus, for W. somnifera the presence of excess calcium in the growing medium is most deleterious compared to other salts. Results also suggest that the nonenzymatic and enzymatic antioxidant systems of both the tissues played a primary role in combating the imposed salt stress.     

Optimization of α‐Amylase Immobilization in Calcium Alginate Beads

Abstract

α‐Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl₂ concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl₂ concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL^?1, and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40°C.     

Purification and properties of the carbonic anhydrase of Rhodospirillum rubrum

The carbonic anhydrase (EC 4.2.1.1) of Rhodospirillum rubrum has been purified to apparent homogeneity and some of its properties have been determined. The enzyme was cytoplasmic and was found only in photosynthetically grown cells. It had a molecular weight of about 28,000, and was apparently composed of two equal subunits. The amino acid composition was similar to that of other reported carbonic anhydrases except that the R. rubrum enzyme contained no arginine. The isoelectric point of the enzyme was 6.2 and the pH optimum was 7.5. It required Zn(II) for stability and enzymatic activity. The K _m(CO₂) was 80 mM. Typical carbonic anhydrase inhibition patterns were found with the R. rubrum enzyme. Strong acetazolamide and sulfanilamide inhibition confirmed the importance of Zn(II) for enzymatic activity as did the anionic inhibitors iodide, and azide. Other inhibitors indicated that histidine, sulfhydryl, lysine and serine residues were important for enzymatic activity.Abbreviation CA carbonic anhydrase In memory of R. Y. Stanier     

The Effect of Various Alkaline Salts on the Glycolate Oxidase of Salicornia europaea and Pisum sativum in vitro

The response of glycolate oxidase from shoots of Salicornia europaea L. and from leaves of Pisum sativum L. to salt treatment during assay was studied by DCPIP reduction and O₂ uptake. In Pisum there was found up to five times more glycolate oxidase activity per gram fresh weight than in Salicornia. However, the calculation of the specific activity pointed out that this result was caused only by the high level of enzyme protein in Pisum, and that specific activity from both species was of equal size. By the DCPIP method it was shown that in test media containing up to 1.0 M NaCl or KCl glycolate oxidase of Salicornia was of equal size compared with the control (medium without additional salts). With 2.0 M NaCl or KCl the activity decreased by about 80 and 30% respectively. Glycolate oxidase of Pisum was somewhat more salt sensitive. 1.0 M NaCl or KCl reduced the activity by about 35%. In the presence of 2.0 M NaCl or KCl the enzyme activity from Pisum was inhibited to about 80 and 60% respectively. By substituting sulfates for chlorides the activity of glycolate oxidase from both Salicornia and Pisum was stimulated strongly. 1.5 M Na₂SO₄ and 0.5 M K₂SO₄ (both are saturated solutions) caused an increase of glycolate activity from Salicornia of about 225 and 185% respectively, and from Pisum of about 50 and 30% respectively. Studying the response of glycolate oxidase to salt treatment by O₂ uptake one must establish that with this method the degree of inhibition of enzyme activity at higher salt concentrations was always more severe than with dye reduction. Addition of 1.0 M NaCl or KCl to the assay medium caused an inhibition of glycolate oxidase activity from Salicornia of about 50% and from Pisum of about 60%. 2.0 M NaCl or KCl reduced the enzyme activity of both Salicornia and Pisum to nearly 10% of control activity. Furthermore, in contrast to DCPIP reduction no stimulating effect of sulfates on glycolate oxidase activity was detectable. Indeed, the inhibitory effect of sulfates was very slight. 1.0 M Na₂SO₄ caused a mean inhibition of glycolate oxidase activity of only 15% with both species, and in the presence of 1.5 M Na₂SO₄ 50% of control activity was measured. At maximal K₂SO₄ concentrations (0.5 M) glycolate oxidase from both Salicornia and Pisum was also unaffected. It is supposed that the described salt tolerance of glycolate oxidase in vitro, possibly is due to an adaptation of the enzyme to high salt levels within peroxisomes in vivo.     

Supplementary CaCl2 ameliorates wheat tolerance to NaCl

Nine-day-old seedlings of two wheat cultivars (Misr1 and Sakha93) were treated with NaCl at 75, 150 and 225 mM for 15 days with or without the presence of 10 mM CaCl₂. All concentrations of NaCl led to significant decreases in fresh and dry weights of only Sakha93; however, Misr1 seemed to be affected only at the highest concentration. Nonetheless, growth parameters of both cultivars under normal conditions were most likely similar. On the other hand, lipid peroxides (as MDA) and H₂O₂ were greatly accumulated particularly in Sakha93; significant increases were detected in Misr1 treated only at 225 mM. Also, all concentrations of NaCl decreased GSH content in Sakha93; nevertheless, there were no great differences among both cultivars under normal conditions. On the other hand, the activities of the enzymatic antioxidants, GR, GST, CAT and POD were unaffected in Misr1 by all concentrations but inhibited in Sakha93. AOX responded differently to NaCl, there were decreases in Misr1 by 75 and 225 mM and in Sakha93 by 75 and 150 mM. However, the application of CaCl₂ alleviated the impacts of NaCl; there was a retraction in growth reduction in Misr1 to reach most likely those of the control. In addition, the accumulated MDA and H₂O₂ were greatly counterbalanced. On the contrary, the decreased GSH contents seemed unrecovered in Sakha93 in spite of the alleviations in magnitudes. Moreover, there were recoveries in the activities of GR and POD in Sakha93; nevertheless, GST and CAT activities remained significantly inhibited. These findings suggest that Misr1 is a more tolerant cultivar to NaCl than Sakha93. Moreover, the results reveal that ROS scavenging is efficient and became more inducible in the less susceptible than in the more susceptible cultivar. The response of AOX appeared to coincide with antioxidants so that the damage which was inflicted by NaCl can be ameliorated by over-expression of antioxidants especially with the presence of CaCl₂.     

Regulation of Neuropeptide-Processing Enzymes by Nitric Oxide in Cultured Astrocytes

Abstract: Nitric oxide (NO), a recently discovered neuro-transmitter, has been shown to have a cytostatic effect on cultured glia. A NO-generating agent, S -nitroso- N -acetyl-penicillamine (SNAP), was used to treat C6 glioma and primary cortical astrocytes. The levels of a monobasic peptide-processing enzyme activity and carboxypeptidase E activity were examined. The cellular levels of these two enzymes are specifically reduced in response to treatment with SNAP. A decrease of ˜30–50% in these two'enzyme activities was seen in both primary astrocytes and C6 glioma cells. This decrease in cellular enzyme activities is not due to increased secretion because the secreted activity is also reduced in response to SNAP treatment in both the glioma cells and the primary astrocytes. Removal of SNAP treatment causes the carboxypeptidase enzyme activity to return to control levels within 3 days. Northern and western blot analyses indicate that the reduced cellular level of carboxypeptidase E is not due to reduced expression of the messenger RNA or protein, suggesting that the SNAP treatment is affecting factors that influence carboxypeptidase E activity. Taken together, these results imply that NO is involved in the regulation of peptide biosynthetic enzymes and this could lead to the antimitogenic action of SNAP on glia.     

Androgen and progesterone metabolism in the central and peripheral nervous system

This paper summarizes the most recent data obtained in the authors' laboratory on the metabolism of testosterone and progesterone in neurons, in the glia, and in neuroblastoma cells. The activities of the 5α-reductase (the enzyme that converts testosterone into dihydrotestosterone, DHT), and of the 3α-hydroxysteroid dehydrogenase (the enzyme that converts DHT into 5α-androstane-3α,17β-diol, 3α-diol) have been first evaluated in primary cultures of neurons, oligodendrocytes and type-1 and -2 astrocytes, obtained from the fetal or neonatal rat brain. All the cultures were used on the fifth day. The formation of DHT or 3α-diol was evaluated incubating the different cultures with labeled testosterone or DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, type-2 astrocytes and oligodendrocytes also possess considerable 5α-reductase activity, while type-1 astrocytes show a much lower enzymatic concentration. A completely different localization was observed for 3α-hydroxysteroid dehydrogenase; the formation of 3α-diol appears to be prevalently, if not exclusively, present in type-1 astrocytes; 3α-diol is formed in very low yields by neurons, type-2 astrocytes and oligodendrocytes. The compartmentalization of two strictly correlated enzymes (5α-reductase and 3α-hydroxysteroid dehydrogenase) in separate central nervous system (CNS) cell populations suggests the simultaneous participation of neurons and glial cells in the 5α-reductive metabolism of testosterone. Subsequently it has been shown that, similarly to what happens when testosterone is used as the substrate, the 5α-reductase which metabolizes progesterone into 5α-pregnane-3,20-dione (DHP) shows a significantly higher activity in neurons than in glial cells; however, type-1 and -2 astrocytes as well as oligodendrocytes also possess some ability to 5α-reduce progesterone. On the other hand, 3α-hydroxysteroid dehydrogenase, the enzyme which converts DHP into 5α-pregnane-3α-ol-20-one, appears to be present mainly in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes show considerable 3α-hydroxysteroid dehydrogenase activity, even if this is statistically lower than that present in type-1 astrocytes. The existence of isoforms of the enzyme involved in androgen and progesterone metabolism is discussed.Finally, the ability of the human neuroblastoma cell line SH-SY5Y to metabolize androgens and progesterone was studied incubating the cells in the presence of labeled testosterone or progesterone to measure, respectively, the formation of DHT or DHP (5α-reductase activity). 3α-Hydroxysteroid dehydrogenase activity was studied by evaluating the conversion of labeled DHT into 3α-diol. The results demonstrate that undifferentiated neuroblastoma cells possess a significant 5α-reductase activity, as shown by the considerable conversion of testosterone into DHT; moreover, this enzymatic activity seems to be significantly stimulated following cell differentiation induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), but not after differentiation induced by retinoic acid (RA). The 5α-reductase present in SH-SY5Y cells is also able to convert progesterone into DHP. In undifferentiated cell, this conversion is about 8 times higher than that of testosterone into DHT. Under the influences of TPA and RA, the formation of DHP follows the same pattern observed for that of DHT. SH-SY5Y cells also appear to possess the enzyme 3α-hydroxysteroid dehydrogenase, since they are able to convert DHT into 3α-diol. This enzymatic activity is not altered following TPA-induced differentiation, and appears to be decreased following treatment with RA. It is suggested that the SH-SY5Y cell line may represent a useful “in vitro” model for the study of the mechanisms involved in the control of androgen and progesterone metabolism in nervous cells.     

Effects of salts on NADH-malate dehydrogenase activity in celery leaves during their maturation and senescence

The effect of several inorganic salts on the activity of NADH-malate dehydrogenase (MDH) from the petiole and the blade of celery leaves of different age, was investigated. KCl and NaCl, in a concentration range of 100–180 mM, activated the enzyme similarly when given in the assay medium. At higher concentrations, both salts became inhibitory. CaCl₂ and MgCl₂ activated the enzyme at a much lower concentration, with MgCl₂ being the weaker activator. The activation of celery leaf MDH by KCl was found to vary with the leaf age. As the leaf matured, the extracted enzyme exhibited increased activation by KCl. However, the transition from maturity to senescence was accompanied by a decline in the activation of the enzyme by the salt. During senescence of detached leave blades in the dark, the MDH activation by KCl undergoes the same changes found in the intact plant. The changes in MDH activity due to either age or KCl, were a result of changes in the V_max and not of the K_m-value for oxaloacetate. Acceleration of senescence by transient water stress or flooding induced a marked increase in the MDH activation by KCl in vitro.It is suggested that the change in the enzyme response to ions is part of a regulation mechanism by which MDH activity is maintained during various developmental stages of the leaf. However, in accelerated senescence situations, this regulation mechanism is disrupted.     

Studies on marine occurring yeasts: Respiration,fermentation and salt tolerance

Summary The effect of various NaCl concentrations on respiration and fermentation rates in cells with or without added glucose as exogenous substrate as well as on respiratory quotients was determined for Debaryomyces hansenii, Saccharomyces cerevisiae, Cryptococcus albidus, and Candida zeylanoides, all yeasts isolated from marine environment. A given strain had about the same respiratory and fermentatory intensity at 0% and 4% NaCl (w/v). A further increase considerably reduced the oxygen uptake or CO₂-evolution. D. hansenii was the most NaCl tolerant yeast tested, giving about 10% activity still at a concentration of 24% NaCl, whether the activities of whole cells or cell homogenates were determined. For S. cerevisiae or Cr. albidus the respiratory activity was reduced to about the same degree at 16% NaCl for whole cells, at 12% NaCl for homogenates of Cr. albidus. A somewhat higher NaCl concentration was evidently tolerated for respiration and fermentation than for growth, very obvious in the case of C. zeylanoides.The minimum values for water activity (a _w) permitting 10% respiration activity were higher when produced by electrolytes (NaCl, KCl, or Na₂SO₄), lower when due to sugars (metabolizable glucose or non-metabolizable lactose) and lowest when due to glycerol. The a _w per se was evidently not solely decisive for the limitation of respiration activity.Attempts were made to assess an effect of high NaCl concentrations on the glucose uptake.The potassium content was higher in cells of the highly halotolerant D. hansenii than in those of the other yeasts and decreased with the increase in external, consequently in internal, Na⁺ concentration. The decrease in K⁺ content can presumably only proceed to a certain extent, below which the ability for growth and respiration was lost.     

Changes in the antioxidative systems of Ocimum basilicum L. (cv. Fine) under different sodium salts

The effects of different sodium salts on some physiological parameters and antioxidant responses were investigated in a medicinal and aromatic plant, Ocimum basilicum L. (cultivar Fine). Plants were subjected to an equimolar concentration of Na₂SO₄ (25?mM) and NaCl (50?mM) for 15 and 30?days. Growth, oxidative stress parameters [electrolyte leakage, peroxidation, and hydrogen peroxide (H₂O₂) concentration], antioxidant enzyme activities [ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2), and peroxidases (POD, EC 1.11.1.7)], as well as antioxidant molecules [ascorbate and glutathione] were determined. The two salts affected leaf growth rates to the same extent, after 15 or 30?days of treatment, indicating a similar effect of Na₂SO₄ and NaCl salinity on growth, even if different (enzymatic and non-enzymatic) antioxidant mechanisms were involved in H₂O₂ detoxification. However, under both salts, the efficiency of the antioxidant metabolism seemed to be sufficient to avoid the deleterious effects of reactive oxygen species (ROS). Indeed, both ion leakage and peroxidation did not change under either Na₂SO₄ or NaCl salinity. As a whole, these data suggest that a cooperative process between the antioxidant systems is important for the tolerance of Ocimum basilicum L., cv. Fine to Na₂SO₄ and NaCl salinity.     

Environmentally controlled changes of phosphoenolpyruvate carboxylases in Mesembryanthemum

NaCl treated Mesembryanthemum crystallinum plants exhibit a Crassulacean acid metabolism. The activity of phosphoenolpyruvate (PEP) carboxylase, the enzyme responsible for CO₂ dark fixation, depends on leaf age showing maximum activity in mature leaves. Electrophoresis revealed that the young leaves possess only two protein bands with PEP carboxylase activity, while older leaves have 3 bands. The removal of NaCl from the soil resulted in the disappearance of the 3rd band obtained after electrophoresis and a decline in the total activity of the PEP carboxylase. The reintroduction of NaCl at the same concentration as before did not restore the activity of the PEP carboxylase nor did it restore the initial electrophoretic band pattern.     

Complexes of glucose oxidase with chitosan and dextran possessing enhanced stability

Abstract

In this study, the different mole ratios of glucose oxidase/chitosan/dextran–aldehyde and glucose oxidase/chitosan/dextran–sulfate complexes were synthesized. The modification of glucose oxidase by non-covalent complexation with dextran and chitosan in different molar ratios was studied in order to increase the enzyme activity. The enzyme/polymer complexes obtained were investigated by UV spectrophotometer and dynamic light scattering. Activity determination of synthesized complexes and free enzyme were performed at a temperature range. The best results were obtained by C_chitosan/C_{dextran–aldehyde} = 10/1 ratio and C_chitosan/C_{dextran–sulfate} = 1/5 ratio that were used in thermal stability, shelf life, salt stress, and ethanol effect experiments. The results demonstrated that both complexes were thermally stable at 60?°C and had superior storage stability compared to the free glucose oxidase. Complexes showed higher enzymatic activity than free enzyme in the organic solvent environment using 10% ethanol. The complexes were resistant to salt stress containing 0.1?M NaCl or CaCl₂. The particle size distribution results of the triple complex evaluated the complexation of the chitosan, dextran derivative, and glucose oxidase. The average size of the triple complex in diameter was found to be 325.8?±?9.3?nm. Overall findings suggest that the complexes of glucose oxidase, chitosan, and dextran showed significant enhancement in the enzyme activity.     

Protective Effects of Resveratrol on Hydrogen Peroxide Induced Toxicity in Primary Cortical Astrocyte Cultures

It is well established that the brain is particularly susceptible to oxidative damage due to its high consumption of oxygen and that astrocytes are involved in a variety of important activities for the nervous system, including a protective role against damage induced by reactive oxygen species (ROS). The use of antioxidant compounds, such as polyphenol resveratrol found in red wine, to improve endogenous antioxidant defenses has been proposed for neural protection. The aim of this study is to evaluate the putative protective effect of resveratrol against acute H₂O₂-induced oxidative stress in astrocyte cultures, evaluating ROS production, glutamate uptake activity, glutathione content and S100B secretion. Our results confirm the ability of resveratrol to counteract oxidative damage caused by H₂O₂, not only by its antioxidant properties, but also through the modulation of important glial functions, particularly improving glutamate uptake activity, increasing glutathione content and stimulating S100B secretion, which all contribute to the functional recovery after brain injury.     

Purification and characterization of a novel extracellular halophilic and organic solvent-tolerant amylopullulanase from the haloarchaeon, Halorubrum sp. strain Ha25

A halophilic archaeon, Halorubrum sp. strain Ha25, produced extracellular halophilic organic solvent-tolerant amylopullulanase. The maximum enzyme production was at high salt concentration, 3–4 M NaCl. Optimum pH and temperature for enzyme production were 7.0 and 40 °C, respectively. Molecular mass of purified enzyme was estimated to be about 140 kDa by SDS–PAGE. This enzyme was active on pullulan and starch as substrates. The apparent K _m for the enzyme activity on pullulan was 4 mg/ml and for soluble starch was 1.8 mg/ml. Optimum temperature for amylolytic and pullulytic activities was 50 °C. Optimum pH for amylolytic activity was 7 and for pullulytic activity was 7.5. This enzyme was active over a wide range of concentrations (0–4.5 M) of NaCl. The effect of organic solvents on the enzyme activities showed that this enzyme was more stable in the presence of non-polar organic solvents than polar solvents. This study is the first report on amylopullulanase production in halophilic bacteria and archaea.     

Stability and Enzymatic Studies of Glucose Dehydrogenase from the Archaeon Haloferax mediterranei in reverse micelles

Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant glucose dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w₀=[H₂O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-glucose to D-glucono-1,5-lactone using NADP⁺ as coenzyme for the glucose dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.     

Carboxyl ester hydrolases production and growth of a halophilic archaeon, Halobacterium sp. NRC-1

The capability of Halobacterium sp. NRC-1 to synthesize carboxyl ester hydrolases was investigated, and the effect of physicochemical conditions on the growth rate and production of esterases was evaluated. The haloarchaeon synthesized a carboxyl ester hydrolase, confirming the genomic prediction. This enzymatic activity was intracellularly produced as a growth-associated metabolite. Esterase activity was assayed using different p-nitrophenyl-esters and triacyl-glycerides, which showed a preference for hydrolyzing tributyrin. The archaeal growth rate and esterase production were significantly influenced by the pH and the NaCl concentration. An interaction effect between temperature and NaCl was also seen. The maximal growth rate and esterase production found for Halobacterium sp. NRC-1 were 0.136 h⁻¹ (at 4.2 M NaCl, pH 6 and 44°C) and 1.64 U/l (at 4.6 M NaCl, pH 6 and 30°C), respectively. Furthermore, the effects of NaCl concentration, pH and temperature on enzyme activity were studied. Two maximal esterase activities were elucidated from the intracellular crude extract when it was incubated at different NaCl concentrations (1 M and 5 M) and at different pHs (6 and 7.5). This is the first report that shows experimentally the synthesis of carboxyl ester hydrolases by Halobacterium sp. NRC-1. This enzyme was found to be extremely halophilic (5 M NaCl) and thermophilic (80°C), making it very interesting for future investigations in non-aqueous biocatalysis.     

The salt-induced dissociation and inactivation of a mammalian enolase: evidence for the formation of active monomers

The gamma gamma isozyme of rabbit enolase was labeled with fluorescein and the effects of NaClO4 on both enzymatic activity and fluorescence polarization were studied. NaClO4, but not NaCl, dissociates and partially inactivates the enzyme. If dissociation is prevented, either by the addition of substrate or by covalently crosslinking the enzyme, inactivation is also prevented. Analysis of the time and concentration dependence of inactivation and dissociation shows that the decrease in activity is a two-step process: D in equilibrium 2M in equilibrium 2M*. Both monomeric forms of the enzyme are catalytically active.