Esterification by immobilized lipase in solvent-free media: kinetic and thermodynamic arguments.

The aim of this study is to characterize, in solvent-free systems (SFS), the kinetic and thermodynamic performance of batch lipase-catalyzed esterification. SFS are compared to a conventional organic solvent, n-hexane. The esterification of oleic acid with ethanol was chosen as a model reaction. The TABEK (thermodynamic activity-based enzyme kinetics) approach was used to rationally analyze kinetics. Influence of the reaction medium on final conversions was also studied. Several factors, such as initial molar ratio of substrates, reactant availability, initial water content, and quantity of immobilized enzyme, were examined. Special attention was also turned to enzyme stability and reuse after reaction, this last item being a prerequisite in the development of industrial processes. SFS proved to be almost as efficient as n-hexane from a kinetic and thermodynamic point of view and offered a better volumetric production.

Water activity control: a way to improve the efficiency of continuous lipase esterification.

During continuous lipase-catalyzed oleic acid esterification by ethanol in n-hexane, the oleic acid conversion, initially at 95%, decreases to 20% after 2 h. This decrease is caused by the accumulation of the water produced in the course of the reaction in the packed-bed reactor (PBR). In order to improve the PBR efficiency, it is necessary to evacuate the water produced. In this study, different approaches have been tested to control the water content in the PBR during continuous esterification. The first approach consisted in improving the water solubility by increasing the reaction medium polarity. The addition of polar additives to n-hexane, the use of more polar solvents, and the use of solvent-free reaction medium were tested as a means to favor the water evacuation from the PBR. First of all, the use ofn-hexane supplemented with acetone (3 M) or 2-methyl-2-propanol (1 M) enabled the conversion to be maintained at higher values than those obtained in pure n-hexane. The replacement of n-hexane by a more polar solvent, like the 5-methyl-2-hexanone, resulted in the same effect. In all cases, conversions at steady-state were always less than 95%, as obtained in pure n-hexane. This is explained by a decrease in the enzyme activity due to the increase in the medium polarity. Nevertheless, an increase in enzyme quantity allowed 90% conversion to be maintained during 1 week using 3 M acetone amended n-hexane. Good results (a steady-state conversion of about 80%) were obtained when esterification was carried out in a solvent-free reaction medium containing 2 M 2-methyl-2-propanol as a polar additive. The second approach consisted in the evaporation of the accumulated water by use of an intermittent airflow. Although this process did not enable constant esterification rate to be maintained, it did enable the initial conversion (95%) to be restored intermittently.

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.

Experimental and theoretical analysis of the chromatographic behaviour of protein purification fusions carrying charged tails.

Poly(glutamic acid) tail consisting of 6 glutamate residues was fused to the N-terminus of Escherichia coli beta-galactosidase (beta-gal), by genetic engineering techniques. The wild-type and modified genes were expressed intracellularly and in soluble state in Escherichia coli, leading to the proteins respectively designated beta-gal2 and E6-beta-gal. Both enzymes were purified by affinity chromatography. The specific activity of purified E6-beta-gal was found to be comparable to the wild-type enzyme and its increased net charge was indicated by lon-Exchange Chromatography (IEC). The use of such a charged fusion for selective recovery of beta-gal from cell extract using IEC and Ion-Exchange Membrane Chromatography (IEMC) was explored. The additional charges enabled the separation factor to be increased about two-fold on both IEC and IEMC, but the IEMC step achieved a better throughput than the IEC step. The selectivity of recovery promoted by the charged tail was further analysed by processing the experimental data obtained in IEC with the Stoichiometric Displacement Model, a recent model very appropriate for the understanding of the retention of polymeric biomolecules on ion-exchangers. It was shown that E6-beta-gal had the same characteristic charge as beta-gal2 but that the binding constant to the ion-exchanger of the tagged beta-gal was 6 times greater than for the wild-type enzyme.

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 reaction-separation process for enzymatic esterification in supercritical carbon dioxide.

The study of enzymatic esterification by an immobilized lipase in supercritical carbon dioxide (SCCO(2)) and in n-hexane, described in our previous works, was extended to continuous operation in a tubular fixed bed. The modeling of the reaction vessel operation was achieved through the use of the simple plug flow model coupled with the appropriate kinetic equation. Comparison with experiments proved to be satisfactory. The study of the postreactional separation, an important feature when using SCCO(2), was undertaken experimentally and good selectivities and product recovery were obtained. (c) 1994 John Wiley & Sons, Inc.

Preparative anion-exchange chromatography of soybean trypsin inhibitor: the alternative of column-overload methods.

The objective of preparative separation is to purify the largest amount of material in the shortest time and at a minimum cost, i.e. to maximize throughout. One of the techniques for increasing throughput is to overload the column while maintaining purity and cycle time at the same level. This principle is applied in sample displacement mode chromatography, in which the column is overloaded with sample mixture until it is completely saturated. Soybean trypsin inhibitor was purified from a crude protein extract by this technique using an analytical anion-exchange column with small particle size (20 microns). The comparison of these results, using the criterion of throughput, with those derived from a conventional scale-up, using a 40-microns preparative column, led to the conclusion that the overloaded 20 microns column gave a higher throughput than the 40-microns column.

Kinetics of lipase-catalyzed esterification in supercritical CO(2).

This study compares two solvents for enzymatic reactions: supercritical carbon dioxide (SCCO(2)) and organic solvent (n-hexane). The model reaction that was chosen was the esterification of oleic acid by ethanol catalyzed by an immobilized lipase from Mucor miehei (Lypozyme). The stability of the enzyme appeared to be quite good and similar in both media but was affected by the water content. Partition of water between solvents and immobilized enzyme has been calculated from experimental adsorption isotherms. The water content of the solid phase has a dramatic influence on the activity of the enzyme and its optimum value for activity was about 10% (w/w) in both media. A kinetic study enabled a Ping-Pong Bi-Bi reaction mechanism with inhibition by ethanol to be suggested. Despite some differences in kinetic constants, activity was in the same range in both media. Hypotheses for explaining the kinetic constant variations have been proposed and particular attention has been paid to the pH effects.

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.

Mechanism of enhanced oxygen transfer in fermentation using emulsified oxygen-vectors.

Limitations of oxygen transfer in fermentation can be solved using auxiliary liquids immiscible in the aqueous phase. The liquids (called oxygen-vectors) used in this study were hydrocarbon (n-dodecane) and perfluorocarbon (forane F66E) in which oxygen is highly soluble (54.9 mg/L in n-dodecane and 118 mg/L in forane F66E at 35 degrees C in contact with air at atmospheric pressure). It has been demonstrated that the use of n-dodecane emulsion in a culture of Aerobacter aerogenes enabled a 3. 5-fold increase of the volumetric oxygen transfer coefficient(k(L)a) calculated on a per-liter aqueous phase basis. The droplet size of the vector played a crucial role in the phenomena. When a static contact between gas bubble and vector droplet was established in water, the vector covered the bubble, in agreement with positive values of the spreading coefficient for these fluids. The determination of the oxygen transfer coefficients (k(L)) in a reactor with a definite interfacial area enabled the main resistance to be located in the boundary layer of the waterside either for a gas-water or a vector-water interface. Because oxygen consumption by weakly hydrophobic cells can only occur in the aqueous phase, the oxygen transfer is achieved according to the following pathway: gas-vector-water-cell. Finally, a mechanism for oxygen transfer within this four-phased system is proposed.

Effect of diffusional resistances on the action pattern of immobilized alpha-amylase.

Alpha-amylase from Aspergillus oryzae has been immobilized onto corn grits and porous silica (specific areas 180 and 440 m2 g-1). Kinetic parameters of immobilized enzyme have been determined. Immobilization of alpha-amylase results in the formation of less polymerized products resulting in an apparent decrease in the number of transglycosylation reactions, for both maltotetraose and starch as substrates, when compared with free enzyme. Diffusional limitations for substrate and products have been quantified in the case of the three supports used. External diffusional resistances were important in all cases for the reaction products, whilst they became negligible for the substrate in the case of silica supports. Moreover, internal transfer limitations were identified with silica 180 m2 g-1 support. It was demonstrated that diffusional resistances were in direct relation to the apparent modification of the enzyme action pattern after immobilization.