Search PubMed⌕ Search

Biomedical subjects

M D Legoy

Publications and source records attributed to M D Legoy.

At least 19 recordsLinked to original sources
PubMed2004

Effects of hydrostatic pressure on horse liver alcohol dehydrogenase (HLADH): a new way of analyzing kinetic study.

Oxidation of ethanol by horse liver alcohol dehydrogenase (HLADH) is monitored under pressure (0.1 MPa - 225 MPa). The pressure-induced modifications of catalytic activity are followed by plotting reaction velocities as a function of substrates concentrations in the traditional double reciprocal form: then, pressure is treated as an activator (p < 100 MPa) or an inhibitor (p<225 MPa). Surprising typical patterns of Lineweaver-Burk curves are observed and interpreted. These results suggest that this approach could be a powerful tool to study enzyme's structure-activity relationship.

Alcohol Dehydrogenase↗
PubMed2001

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system obeys a Ping Pong Bi Bi mechanism with competitive inhibition by the alcohol substrate and water.

The kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by Candida antarctica lipase B supported onto silanized Chromosorb P was studied in a continuous solid/gas reactor. In this system the solid phase is composed of a packed enzymatic sample and is percolated by nitrogen as carrier gas, which simultaneously carries substrates to the enzyme while removing reaction products. In this reactor the thermodynamic activity of substrates and effectors can be perfectly adjusted allowing kinetic studies to be performed under different operating conditions. The kinetics obtained for alcoholysis were suggested to fit a Ping Pong Bi Bi mechanism with dead-end inhibition by the alcohol. The values of all apparent kinetic parameters were calculated and the apparent dissociation constant of enzyme for gaseous ester was found very low compared with the one obtained for liquid ester in organic medium, certainly due to the more efficient diffusion in the gaseous phase. The effect of water thermodynamic activity was also investigated. Water was found to act as a competitive inhibitor, with a higher inhibition constant than n-propanol. Thus alcoholysis of gaseous methyl propionate and n-propanol catalyzed by C. antarctica lipase B was found to obey the same kinetic mechanism as in other non-conventional media such as organic liquid media and supercritical carbon dioxide, but with much higher affinity for the substrates.

1-Propanol↗
PubMed2001

Gas phase biotransformation reaction catalyzed by baker's yeast.

The gas phase continuous production of acetaldehyde from ethanol and hexanol from hexanal using dried baker's yeast was studied as an alternative approach to conventional processes. The effects of water activity, activity of substrates, and amount of yeast on the performance of the continuous bioreactor were investigated. The extent of yeast hydration and ethanol activity are the most important factors affecting yeast activity and stability.

Acetaldehyde↗
PubMed2001

Identification of an indigo precursor from leaves of Isatis tinctoria (Woad).

Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we describe an HPLC method dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and isoindirubin) and indigo precursors produced in I. tinctoria (Woad). This work, carried out with two cultivars of I. tinctoria, has confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-ketogluconate) and indican (indoxyl-beta-D-glucoside). We suggest the name "isatan C" for this new indigo precursor in I. tinctoria. Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan C reacts with isatan B increasing the red pigment production.

Brassicaceae↗
PubMed1997

The kinetic behaviour of a two-enzyme system in biphasic media: coupling hydrolysis and lipoxygenation.

Analysis of the kinetic behaviour of a two-enzyme-system carrying out two consecutive reactions was investigated in macroheterogeneous biphasic media (octane/buffer pH 9.6, v/v = 1:1). The lipase-catalysed hydrolysis of trilinolein and the subsequent lipoxygenation of the liberated linoleic acid, were coupled in a modified Lewis cell with a well-defined liquid/liquid interfacial area. Trilinolein was dissolved in the organic phase and hydrolysed in the presence of Mucor javanicus lipase at the organic/aqueous interface. Linoleic acid, liberated after hydrolysis was transferred to the aqueous phase and reacted with lipoxygenase. This reaction consumed linoleic acid and produced hydroperoxides, which favoured the transfer of residual linoleic acid, since they possess surface active properties. Catalysis and transfer influenced each other reciprocally. At low substrate concentrations, cooperativity phenomena were observed in the experimental and also the modelled two-enzyme systems. When the initial substrate concentration was high, the kinetic behaviour of the two-enzyme system in a compartmentalised medium, seemed to be independent of the substrate concentration, unlike that observed in homogeneous monophasic enzymology. The numerical integration program used to model the two-enzyme system was based on results obtained in separate studies of the following three phenomena: (1) trilinolein hydrolysis in biphasic medium. (2) linoleic acid transfer across a liquid/liquid interface and (3) lipoxygenation in an aqueous media. Results obtained by modelling were similar to the results observed experimentally.

Hydrolysis↗
PubMed1996

The pressure-dependence of two beta-glucosidases with respect to their thermostability.

A comparative study of temperature and pressure effects were carried out by using two homologous enzymes exhibiting different thermostability and oligomery: almond beta-glucosidase and Sulfolobus solfataricus beta-glucosidase. Both the activity and stability were studied using an in-house built bioreactor allowing injection, stirring, sampling and on-line spectrophometric monitoring with retention of pressure up to 2.5 kbar and temperature control possible up to 150 degrees C. Almond beta-glucosidase, the most pressure sensitive enzyme of the two was continuously affected by pressure up to 1.5 kbar. Activation volume changes revealed that the inactivation of almond beta-glucosidase was due to both catalytic step inactivation and enzyme-substrate binding inactivation. Structural modifications generated by pressure, related to a loss of activity did not affect the global conformation of almond beta-glucosidase, after depressurization. In contrast, Sulfolobus solfataricus beta-glucosidase was a highly barostable enzyme. It maintained a half-life of 91 h at 60 degrees C and 2.5 kbar. Moreover, this enzyme appeared to be activated by pressure between atmospheric pressure and 2.5 kbar with a maximal activity at 1.25 kbar. However, this enzyme still displayed the best catalytic efficiency at atmospheric pressure because of a Km value drastically increased by pressure. Activation volume changes indicated that the hydrolysis reaction catalysed by Sulfolobus solfataricus beta-glucosidase, was alternatively favoured and disfavoured by pressure due to the catalytic step activation or inactivation associated with the enzyme-substrate binding step being continuously inactivated by pressure.

Copper↗
PubMed1996

Hydrostatic pressure induces conformational and catalytic changes on two alcohol dehydrogenases but no oligomeric dissociation.

A comparison between the pressure effects on the catalysis of Thermoanaerobium brockii alcohol dehydrogenase (TBADH: a thermostable tetrameric enzyme) and yeast alcohol dehydrogenase (YADH: a mesostable tetrameric enzyme) revealed a different behaviour. YADH activity is continuously inhibited by an increase of pressure, whereas YADH affinity seems less sensitive to pressure. TBADH activity is enhanced by pressure up to 100 MPa. TBADH affinity for alcoholic substrates increases if pressure increases, was TBADH affinity for NADP decreases when pressure increases. Hypothesis has been raised concerning the dissociation of oligomeric enzymes under high hydrostatic pressure ( < 200 MPa) [1]. But in the case of these two enzymes, unless the oligomers reassociate very quickly (< 1 min), the activity inhibition of YADH at all pressures and TBADH for pressures above 100 MPa is not correlated to subunit dissociation. Hence we suggest that enzymes under pressure encounter a molecular rearrangement which can either have a positive or a negative effect on activity. Finally, we have observed that the catalytic behaviour of alcohol dehydrogenases under pressure is connected to their thermostability.

1-Propanol↗
PubMed1993

Biocatalysis in the gas phase.

The biocatalysis of substrates in the gas phase may offer advantages over many conventional solution-based reactions, both in analytical devices and in bioreactors designed to accommodate this new technology. To date, however, the range of substrates for which gas-phase biocatalysis has been shown to be suitable is limited. Further research is required to establish the parameters that affect the kinetics and productivity of such systems.

Biotechnology↗
PubMed1991

Modulation of lipase hydrolysis and synthesis reactions using carbohydrates.

A novel method for modulation of lipase hydrolysis and synthesis lipase was investigated by using carbohydrates in the microenvironment of the Candida rugosa enzyme. The influence of the addition of different sugars to the previously dialysed enzyme was tested on the two reactions. Rates of hydrolysis were lowered by using dialysed enzyme but were increased after sugar addition, regardless of the identity of the added sugar. In contrast, synthesis reaction rates depended on the nature of the carbohydrate. Rates were increased by adding lactose, which is not a water activity depressor, but were lowered by adding fructose, glucose, sucrose or sorbitol, which are all water activity depressors.

Candida↗
PubMed1990

The control of lipase-catalysed transesterification and esterification reaction rates. Effects of substrate polarity, water activity and water molecules on enzyme activity.

The reaction rate of two lipase-catalysed reactions, esterification and transesterification, were studied in a liquid/solid two-phase system in order to investigate the effect of water partition between the enzyme preparation and the liquid phase composed of only the reactants, i.e. without the conventional solvents. Lipase from Candida cylindracea was used for these studies. The enzyme was inactive in dehydrated systems. In the case of monoester synthesis, the reaction rate increased with increasing water activity. The reaction rates of the non-specific C. cylindracea lipase-catalysed reactions were very sensitive to the nature of the substrates in this unusual system. For instance, the transesterification reaction rate of ethyl propionate was 48 times higher with nonanol than heptanol in the case of dehydrated substrates, but only 2.2 times higher in the case of water-saturated substrates. The results presented here demonstrate the absolute necessity to consider the polarity of every substrate, because of its ability to modify the water partition between the solid phase (enzyme preparation) and the liquid phase (substrate and product), which results in drastic changes in enzyme activity. Contrary to esterification, which is known to be activated by the water produced, the rate of transesterification remained constant at the beginning of the reaction. However, when transesterification and esterification were carried out in the same liquid phase, the transesterification reaction rate was controlled by the water produced by the concomitant esterification. Activation effects of the water molecules produced during the enzymatic reaction were of exactly the same order of magnitude for both reactions.

Alcohols↗
PubMed1990

Phospholipid synthesis by extracellular phospholipase A2 in organic solvents.

The catalytic activity of extracellular phospholipase A2 was studied in low polarity solvents where hydrolytic enzymes have been demonstrated to catalyze synthesis reactions. It was demonstrated that extracellular phospholipase A2 can catalyze the esterification of lysophosphatidylcholine with oleic acid. Up to 6.5% of lysophosphatidylcholine can be esterified into phosphatidylcholine. This activity requires a preincubation of the enzyme in a pH 9 aqueous solution containing calcium, before the incubation in the non-aqueous solvent. No transfer of fatty acid between a phospholipid and a lysophospholipid or between two phospholipids was observed. These results may be useful in understanding the function of the membrane phospholipase A2 which may catalyze acylation or deacylation depending on the local physico-chemical environment.

Calcium↗
PubMed1990

Water activity as a key parameter of synthesis reactions: the example of lipase in biphasic (liquid/solid) media.

Ester synthesis catalyzed by Candida cylindracea lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was investigated in solid/liquid biphasic media containing the enzyme preparation and reactants without addition of organic solvents not participating in the reaction. Although the effects of water on enzyme kinetics have been abundantly studied in nearly anhydrous media, reactions in which water is produced have not been investigated. The effect of water produced by the reaction itself on the enzymatic activity was studied. The dispersion of water in a shaken, nearly anhydrous medium was shown to be responsible for the lack of activity of the enzyme. In contrast, when slowly shaken, the enzyme was fully activated by the water furnished as a product of the reaction. However, when experiments were performed in a two-phase aqueous/organic system with previously solubilized enzyme in water, the enzyme activity was increased by shaking and was of the same order of magnitude as in nearly anhydrous media. Under low water activity conditions, a powerful agitation can lead to slower reaction rate, because water, a product of esterification, is not retained in the microenvironment of the enzyme to activate it. The activation effect of water produced by the reaction was clearly shown using enzyme preparations shaken in an anhydrous medium and previously equilibrated at low water activities (aw = 0.13 and 0.69). This activation did not occur for an enzyme preparation equilibrated at high aw (0.89) or for a preparation gently shaken in a water-saturated medium. The lag time preceding activation of the enzyme increased with the extent of enzyme dehydration.(ABSTRACT TRUNCATED AT 250 WORDS)

Candida↗
PubMed1989

Dynamic interactions between enzyme activity and the microstructured environment.

A new approach for the study of an enzyme's relationship with its own reaction medium has been developed. One technique of micellar enzymology is the use of pseudohomogeneous systems composed of surfactant/water/organic solvent. In such systems, the physicochemical properties and textures of the medium depend on the relative ratios of the different components. Enzymes are catalytically active in such systems and up to the present have been studied in different microenvironments, such as micelles, microemulsions and lyotropic liquid crystals. Our purpose was to develop a system in which the enzyme could, by its activity, modify one of the components in such a way that the relative ratios among them changed sufficiently to produce a transition from one phase domain to another. The three components, water (or glucose in water), octanol and octyl-beta-D-glucoside, form a classical ternary water/oil/surfactant system. The relevant phase diagram shows different macroheterogeneous phases and microstructured domains. The enzyme beta-D-glucosidase hydrolyses octyl-beta-D-glucoside to form glucose and octanol. The enzyme was found to change the relative ratios of water (or glucose in water), octanol and octyl-beta-D-glucoside in such a manner that the physicochemical structure of the medium was modified. At the beginning of the reaction beta-D-glucosidase was present in a micellar solution of octyl-beta-D-glucoside in water. As the enzymatic reaction proceeded, the medium became biphasic. One of the two phases was the micellar solution of octyl beta-D-glucoside in water, while the other phase was either a microemulsion or a liquid crystalline phase. In addition the enzyme, through its catalytic activity, was able to modify the physiocochemical properties of the reaction medium.

Chemical Phenomena↗
PubMed1988

Enzymatic ester synthesis with continuous measurement of water activity.

Ester synthesis from aliphatic monoalcohols and organic acids was investigated by using a microbial lipase. The reaction medium only contained the substrates and the enzyme without addition of water or organic solvent. During the reaction, water was produced and the water activity (aw) increased. Batch reactors and continuous-flow reactors were used. In batch, the aw was 0.13 at the beginning of the reaction and increased to reach a plateau at 0.77, after which ester synthesis continued without modification of the aw. Different alcohols and acids were tried in solid-liquid reactors, and all cases synthesis occurred, leading to a significant increase in the water activity. For continuous-flow reactors, the use of silica beads retaining water inside the reactor where the enzymatic reaction took place resulted in some control of the enzymatic reaction by changing the aw.

Alcohols↗
PubMed1987

Interesterification and synthesis by Candida cylindracea lipase in microemulsions.

Unusual reactions of interesterification and synthesis catalyzed by Candida cylindracea lipase have been tested in reverse microemulsions. The microemulsions used are made of fatty acids or triglycerides, the enzyme dissolved in a very low water quantity, Brij 35 used as surfactant and an alcoholic cosurfactant. In such a system, fats and alcohols are both the substrates of the enzyme and the microemulsion components. Incidentally, non specific Candida cylindracea lipase does not catalyze interesterification of short chain triglycerides, revealing a specificity for the chain length. Interesterification reactions tested in the presence of a given water quantity but with varying water activities show that it is the water activity and not the water quantity which is a fundamental parameter of the system. The effect of the surfactant (Brij 35) on the interesterification reaction is studied. Heptyl-oleate synthesis catalyzed by non-specific lipase is obtained in microemulsions at a 98% yield. Synthesis of glycerol esters is also tested in monophasic medium and mono and diglycerides are obtained.

Candida↗