Biomedical subjects
Publications and source records attributed to M D Legoy.
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.
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.
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.
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.
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)
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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.
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.
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.
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3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50) from Pseudomonas testosterone was inactivated by superoxide radicals generated by the aerobic xanthine oxidase reaction. Superoxide dismutase, NAD+, bovine serum albumin and histidine and cysteine as free amino acids partially protected the enzyme from inactivation. NADH-binding properties were determined by fluorescence spectroscopy, and no variation was found between native enzyme and the unmodified fraction of the partly inactivated one. The fluorescence emission maximum for the completely inactivated enzyme was shifted 10 nm to a longer wavelength when compared with the native one, and it seems possible that the modification of histidine and cysteine residues by superoxide radicals causes the conformational change of the enzyme and the consequent loss of catalytic activity.
3 beta, 17 beta-Hydroxysteroid dehydrogenase (3 beta 17 beta HSDH) is an NAD-dependent dehydrogenase which has a double specificity for the 3- and 17-positions on the steroid skeleton. When dehydroepiandrosterone (DHEA) is used as steroid substrate, and the assay coupled with ketosteroid-isomerase, the two reactions occur alternately and each reaction on the 3-position produces a chromophoric molecule. These two reactions can follow one another without dissociation of the coenzyme from the enzyme binding site. This is confirmed by competition experiments with another dehydrogenase.
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Two biological approaches have been investigated for specific modifications of steroids. The first one uses the purified enzyme for the specific dehydrogenation of androsterone to androstanedione. The enzyme used is 3 alpha-hydroxysteroid dehydrogenase which requires a cofactor (NAD). A cofactor regeneration is needed so that the process could work continuously. The conjugation of two points (immobilization of the enzyme and optimization of the ratio methanol-water) allows a continuous work of the enzyme during 25 days. Moreover, we propose a chemical regeneration of the cofactor using methoxy derivative of phenazine methosulphate. Right now it is the limiting step of the process. The second approach of steroid modification uses a whole mycelium of Aspergillus phoenicis for the specific hydroxylation of progesterone to 11 alpha-hydroxy progesterone. The transformation of 90% of the progesterone is obtained with calcium alginate immobilization and the lowest number of products is obtained at pH lower than 2.5 with carrageenan and polyurethane immobilization. It seems promising to apply immobilized biocatalysts to the bioconversion of hydrophobic compounds in organic solvents system.
A functional NAD molecule was immobilized at the active site of Alcohol dehydrogenase within a proteic membrane. The presence and the functionality of the cofactor was checked by fluorescence analysis. The dehydrogenase NAD membrane does not require addition of soluble cofactor for its activity. The system represents a new worthwhile approach because both problems of retention and regeneration of cofactor are solved. The method can be used not only for industrial and analytical applications but also to try to get a better understanding of the kinetics and mechanisms of the catalytic action of dehydrogenase.