Sylvain Lamare

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.

A Water Molecule in the Stereospecificity Pocket of Candida Antarctica Lipase B Enhances Enantioselectivity towards Pentan‐2‐ol

The effect of water activity on enzyme‐catalyzed enantioselective transesterification was studied by using a solid/gas reactor. The experimental results were compared with predictions from molecular modelling. The system studied was the esterification of pentan‐2‐ol with methylpropanoate as acyl donor and lipase B from Candida antarctica as catalyst. The data showed a pronounced water‐activity effect on both reaction rate and enantioselectivity. The enantioselectivity increased from 100, at water activity close to zero, to a maximum of 320, at a water activity of 0.2. Molecular modelling revealed how a water molecule could bind in the active site and obstruct the binding of the slowly reacting enantiomer. Measurements of enantioselectivity at different water‐activity values and temperatures showed that the water molecule had a high affinity for the stereospecificity pocket of the active site with a binding energy of 9 kJ mol−1, and that it lost all its degrees of rotation, corresponding to an entropic energy of 37 J mol−1 K−1.

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.

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system: effects of water on kinetic parameters.

The influence of water on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates to the enzyme while removing reaction products. In this system, interactions between the enzyme and nonreacting molecules are avoided, since no solvent is present, and it is thus more easy to assess the role of water. To this end, alcohol inhibition constant, substrates dissociation constants as well as acylation rate constant and ratio of acylation to deacylation rate constants have been determined as a function of water activity (a(w)). Data obtained highlight that n-propanol inhibition constant and dissociation constant of methyl propionate are a lot affected by a(w) variations whereas water has no significant effect on the catalytic acylation step nor on the ratio of acylation to deacylation rate constants. These results suggest the water-independent character of the transition step.

An activity, stability and selectivity comparison of propioin synthesis by thiamine diphosphate-dependent enzymes in a solid/gas bioreactor.

Enzymatic carboligation in a solid/gas bioreactor represents a new challenge in biotechnology. In this paper, the continuous gas‐phase production of propioin from two propanal molecules by using thiamine diphosphate‐dependent enzymes was studied. Two enzymes were used, namely benzaldehyde lyase (BAL) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD) from Pseudomonas putida. The enzymes are homologous and catalyze carboligase and carbolyase reactions in which no external cofactor regeneration is needed. The influence of water and substrate activity on the initial reaction rate and biocatalyst stability was investigated. An increase in water activity raised the initial reaction rates to the maximal values of 250 and 80 U g−1 for BAL and BFD, respectively. The half‐life showed the same trend with maximal values of 50 and 78 min for BAL and BFD, respectively. The increase in the half‐life by increasing water activity was unexpected. It was also observed that BFD is more stable than BAL in the presence of the substrate propanal. Both enzymes showed substrate inhibition in the kinetic studies, and BAL was also deactivated during the reaction. Unexpectedly, the stereoselectivity of both enzymes (ee of 19 % for BAL and racemic mixture for BFD) was significantly impaired in the gas phase compared to the liquid phase.

Water plays a different role on activation thermodynamic parameters of alcoholysis reaction catalyzed by Lipase in gaseous and organic media

The effect of water on the alcoholysis of methyl propionate and n-propanol catalyzed by immobilized Candida antarctica lipase B (CALB) has been compared in a continuous solid-gas reactor and in an organic liquid medium. The enthalpic and entropic contributions of water to the Gibbs free energy of activation in the gas phase were different from the ones in the organic phase, the inverse trends being observed for the variation of both DeltaH* and DeltaS* with water activity. Different phenomena were identified for their influence on the thermodynamic parameters. When increasing a(w), the enhanced flexibility of the enzyme was predominant in the gas phase whereas substrate-solvent interactions due to an increased polarity of the solvent affected mainly the thermodynamic parameters in the organic phase. The observed variations of DeltaG* with water activity were in accordance with kinetics results previously obtained in both reaction media.