Jean-Stéphane Condoret

Kinetic study of esterification by immobilized lipase in n-hexane

The kinetic of the esterification of oleic acid by ethanol catalyzed by immobilized lipase of Mucor miehei in n‐hexane as a solvent has been completely studied. The kinetics of the reaction are suggested to agree with a Ping‐Pong Bi Bi mechanism in which only inhibition by excess of ethanol has been identified. Values of all apparent kinetic parameters were computed. No evidence of any significant external diffusional limitation which could account for these values has been detected. Optimization of water content through distribution ratio of water between solvent and support was examined.

Kinetics of geranyl acetate synthesis by lipase-catalysed transesterification in n-hexane

The synthesis of geranyl acetate by transesterification catalysed by immobilized lipase of Mucor miehei was studied. To obtain the highest yield for geranyl acetate synthesis, the effect of ester chain length on the rate of reaction was investigated. Propyl acetate was found to be the best substrate for geranyl acetate synthesis. The kinetics of reaction are suggested to agree with a ping-pong Bi Bi mechanism in which inhibition by excess of geraniol has been identified. The values of all apparent kinetic parameters were computed. No evidence of any significant external diffusional limitation that could account for these values has been detected. In order to define the optimal conditions of the reaction, optimization of water content through the distribution ratio of water between solvent and support was examined.

Geranyl acetate synthesis by lipase-catalyzed transesterification in supercritical carbon dioxide

The synthesis of geranyl acetate by transesterification catalyzed by immobilized lipase from Mucor miehei was studied in supercritical carbon dioxide (SCCO2) and compared with n-hexane. The influence of water content on the thermostability of the enzyme was investigated in SCCO2. The increase of water content and increase of temperature diminished enzyme stability. The half-life of the dry enzyme in SCCO2 at 100°C was about 24 h. The effect of ester chain length on the rate of reaction was investigated. Propyl acetate was found to be the best substrate for geranyl acetate synthesis in both media. In order to define the optimal conditions of the reaction, optimization of water content was carried out. The water content of the solid phase seemed to be the most important factor for the activity of the enzyme. The same optimum, 8–10% (w/w) water content, was determined in both media. The kinetics of the reaction are suggested to fit a Ping-Pong Bi Bi mechanism in which inhibition by excess of geraniol has been identified. The values of all apparent kinetic parameters which were not affected by the external diffusional limitations were calculated. The differences between the kinetic constants in both media were discussed. Considering the reaction rates and the maximum velocity of the reaction, the SCCO2 is not better than the conventional organic solvents, such as hexane, for the reaction of transesterification between geraniol and propyl acetate.

Comparison of lipase-catalysed esterification in supercritical carbon dioxide and in n-hexane

SummaryOleic acid esterification by ethanol has been performed by an immobilized lipase fromMucor miehei in supercritical carbon dioxide and in n-hexane as solvents. In both media, determination of apparent kinetic constants has been achieved and influence of water content has been shown to be different due to various rates of water solubilities. Stability of the lipase has been proved to be correct and similar in both solvents. Inhibition by ethanol excess has been found but is greater in n-hexane. That can explain the higher initial velocities obtained in supercritical carbon dioxide for the highest ethanol concentrations.

Enzymes in non-conventional phases

The use of enzymes for technical applications has become increasingly important in different areas of biotechnology such as the food or pharmaceutical industries. Various processes have been developed using soluble or immobilized enzymes mainly in aqueous reaction phases. However, the catalytic activity of enzymes not only in water, but also in other solvents was first investigated at the beginning of the century (Bourquelot, E. and Bridel, M., 1911). Apart from water as the exclusive solvent and reaction phase, many kinds of solvent systems for enzymatic reactions became the subject of intensive research over the last 10-15 years (Buckland, Dunnill and Lilly, 1975; Klibanov, Samokhin, Martinek and Berezin, 1977; Nakanishi and Matsuno, 1986; Chen and Sih, 1989; Klibanov, 1990; Blanch, 1992; Carrea et al., 1992).

Use of supercritical CO2 for bone delipidation

Supercritical carbon dioxide was used for bone delipidation. It appeared that this technology is very efficient since supercritical CO2 is able to diffuse into microporous solids much better than liquids and that it has a good solvent capacity for lipids. This extraction is the ideal first step of any bone processing because microporosity of bone tissue becomes much more accessible, which may enhance osteoconduction once implanted. Moreover, it is safe since it involves no toxic chemical and is potentially usable with allografts as well as xenografts.

Continuous operation of lipase-catalyzed reactions in nonaqueous solvents : Influence of the production of hydrophilic compounds

In the field of biocatalysis in nonaqueous media, water has been identified as a crucial parameter which has to be carefully controlled. This article studies the continuous operation of a water-producing enzymatic reaction, here the esterification of oleic acid by ethanol in n-hexane catalyzed by Lipozyme(TM). The conversion decreased significantly over time, eventually coming to a lower steady-state level. This would be due to the accumulation of the produced water into the enzyme fixed-bed reactor, n-hexane being unable to evacuate this water out of the reaction vessel, because of the low polarity of this solvent. Therefore the conversion decreased until the produced water could be eliminated by the solvent achieving a steady state with a lower conversion. In supercritical carbon dioxide, a more hydrophilic solvent, steady state is at once obtained. This approach has been extended to reaction producing a hydrophilic compound, here glycerol during the transesterification between triolein and ethanol, and similar conclusions can be made. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 232-237, 1997.

Extractive fermentation of aroma with supercritical CO2

This work deals with the feasibility of achieving an extractive fermentation of 2-phenylethyl alcohol, the rose aroma, coupling fermentation with Kluyveromyces marxianus and supercritical carbon dioxide (SCCO2) extraction. The extractive process is, in this case, of special interest due to the strong yeast inhibition by 2-phenylethyl alcohol. First results confirmed that direct SCCO2 extraction is not possible, due to a drastic CO2 effect on cell viability. It is therefore necessary to perform cell separation prior to the extraction. Aroma extraction conditions from a synthetic mixture were then optimized, a pressure of 200 bar and a temperature in the range 35-45 degrees C being chosen. Under these conditions, the distribution coefficient Kd is 2 times higher than during the extraction using a conventional organic solvent, n-hexane. Using a simple model of aroma partition between aqueous and SCCO2 phases, the parameters of a continuous extraction from a synthetic broth were defined. The two substrates, glucose and phenylalanine, are not extracted whatever the conditions. As predicted by the model, more than 90% of 2-phenylethyl alcohol can be extracted, while the extraction of ethanol, the second main product, can be easily tuned with respect to operating conditions, as a function of its influence on the fermentation. Finally, the feasibility of the aroma recovery using two depressurization steps at the outflow of the extraction vessel was demonstrated; 97% of the extracted aroma was recovered, and a mass purity of 91% was achieved. Copyright 1999 John Wiley & Sons, Inc.

Fatty Acid Esterification in Supercritical Carbon Dioxide

Abstract Enzymatic synthesis of ethyloleate, from oleic acid and ethanol, catalyzed by an immobilized lipase from Mucor miehei , has been performed in supercritical carbon dioxide (SCCO 2 ) and in n-hexane. Batch studies of initial velocities have allowed the investigation of protein stability, influence of water content and the reactional mechanism. A continuous packed bed reactor has been modelized, after generalization of the initial velocity equation, using a plug-flow reactor model. Finally, the complete reaction-separation process with recycling of the solvent has been examined in a small pilot plant with four post reactional separators.