Marie-Dominique Legoy

Microwave radiation can increase the rate of enzyme-catalysed reactions in organic media

Abstract Irradiating a hydrated lipase enzyme suspended in organic media using microwaves (2.45 GHz, 50°C) enhanced the reaction rate by 2–3 fold over classical heating and the apparent non-thermal effects observed were dependent on the hydration state of the enzyme in the organic medium.

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.

Stability improvement of immobilized Candida antarctica lipase B in an organic medium under microwave radiation

The influence of microwave heating on the stability of immobilized Candida antarctica lipase B was studied at 100 degrees in an organic medium. The microwave radiation was carried out before enzymatic reaction (storage conditions) or during the enzymatic catalysis (use conditions). In both cases, enzymatic stability was higher under microwave heating than under conventional thermal heating, in strictly identical operating conditions. Furthermore, the gain of enzymatic stability under microwave heating appears to be higher in a more polar solvent, which interacts strongly with the microwave field. Our results suggest that microwave radiation has an effect, not related to temperature, on the process of enzymatic inactivation.

Influence of microwave irradiation on enzymatic properties: applications in enzyme chemistry

Although microwave-assisted reactions are widely applied in various domains of organic chemistry, their use in the area of enzyme chemistry has been rather limited, due to the high temperatures associated with the microwave heating: Because current technology, allows a good control of reaction parameters, several examples of microwave-assisted enzyme chemistry have been reported, using stable and effective biocatalysts (modified enzymes). The purpose of this review is to highlight the applications and studies on the influence of microwave irradiation on enzymatic properties and their application in enzyme chemistry.

Influence of microwave radiation on free Candida antarctica lipase B activity and stability

The influence of microwave heating on free Candida antarctica lipase B activity and stability was studied over the temperature range from 40 to 110 degrees C. Concerning the lipase activity, identical initial rate and conversion yield were obtained under microwave radiation and classical thermal heating for the alcoholysis between ethyl butyrate and butanol in a solvent-free system. On the other hand, the kinetics of the free lipase inactivation in butanol appears to be influenced by the heating mode. The Arrhenius plot obtained under classical heating was linear over all the temperature range studied whereas a biphasic Arrhenius plot was obtained under microwaves. The non-classical effect of the microwave heating on the initial rate of the enzymatic inactivation was thus dependent on the temperature of incubation.

Solid/gas bioreactors: powerful tools for fundamental research and efficient technology for industrial applications

Solid–gas biocatalysis appears today to be a promising technology for fundamental research and for the development of new cleaner industrial processes. The use of enzymes or whole cells at the solid–gas interface now appears concurrent to liquid processes and presents some very interesting features since total thermodynamic control of the system can be achieved easily. Moreover, from a technological point of view, solid–gas systems offer very high production rates for minimal plant sizes, allow important reduction of treated volumes and permit simplified downstream processes. These advantages result from the ability to precisely control all the thermodynamic parameters influencing not only the kinetics of the reactions performed, but also the stability of the biocatalysts working with biological catalysts at elevated temperatures. In this article, an overview of some existing systems and application of solid–gas technology to fundamental studies related to the influence of the microenvironment on biocatalysts is given. The potential of this peculiar system, and examples of applications that should benefit from the technology are presented herein.

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.

Expeditious routes to 4-alkoxyquinazoline-2-carbonitriles and thiocarbamates via N-arylimino-1,2,3-dithiazoles using microwave irradiation☆

Abstract Conversion of N -arylimino-4-chloro-5 H -1,2,3-dithiazole 11 into the 4-alkoxyquinazoline-2-carbonitriles 13a-i and of the aryl isothiocyanates 15 into aryl thiocarbamates 16a-j with sodium alkoxides in the corresponding alcohol, either by conventional thermolysis or by microwave irradiation are described and directly compared. Microwave irradiation of the solutions in open vessels in a monomode system with focused irradiation and continuous temperature control (Synthewave S402 reactor) usually gave cleaner, faster and higher yielding reactions. These reactions could be safely and beneficially scaled up to multigram quantities in a larger reactor (Synthewave S1000).

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.

Selective enzymic esterification of free fatty acids with n-butanol under microwave irradiation and under classical heating

Selective enzymic esterification of free fatty acids, obtained from blackcurrant oil by chemical saponification, with n-butanol using four immobilized lipases under microwave irradiation and under classical heating was studied. A positive effect of microwave irradiation on chemical yields of the products of the enzymic reactions and specificity of lipases were observed in comparison with a controlled heating in an incubator equipped with shaking (classical heating) applied during the identical enzyme-mediated processes. The maximum quantity of γ-linolenic acid (30%) was obtained with Lipozyme used as biocatalyst of the reaction under microwave irradiation. The maximum quantity of butyl γ-linolenate (20%) was obtained by a Pseudomonas cepacia lipase catalyzed esterification under classical heating.