A. van der Padt

The effect of organic solvents on the equilibrium position of enzymatic acylglycerol synthesis

The effect of organic solvents on the equilibrium position of lipase‐catalyzed esterification of glycerol and decanoic acid has been investigated. The reaction is carried out in an aqueous‐organic two‐phase system. In polar solvents, high mole fractions of monoacylglycerol and low mole fractions of triacylglycerol and measured, while in nonpolar solvents, the measured differences in the mole fractions of monodi‐, and triacylglycerols are less. There is a good correlation between the ester mole fractions at equilibrium and the log P of the solvent (partition coefficient in n‐octanolwater), however, only if the group of tertiary alcohols is excluded. In the plot of the easter mole fractions as a function of the logarithm of hte solubility of water in the organic solvent, the tertiary alcohols can be included; however, in this case other deviations appear.

Modelling and parameter estimation of the enzymatic synthesis of oligosaccharides by beta-galactosidase from bacillus circulans

The aim of this research is to develop a model to describe oligosaccharide synthesis and simultaneously lactose hydrolysis. Model A (engineering approach) and model B (biochemical approach) were used to describe the data obtained in batch experiments with beta-galactosidase from Bacillus circulans at various initial lactose concentrations (from 0.19 to 0.59 mol.kg(-1)). A procedure was developed to fit the model parameters and to select the most suitable model. The procedure can also be used for other kinetically controlled reactions. Each experiment was considered as an independent estimation of the model parameters, and consequently, model parameters were fitted to each experiment separately. Estimation of the parameters per experiment preserved the time dependence of the measurements and yielded independent sets of parameters. The next step was to study by ordinary regression methods whether parameters were constant under the altering conditions examined. Throughout all experiments, the parameters of model B did not show a trend upon the initial lactose concentration when inhibition was included. Therefore model B, a galactosyl-enzyme complex-based model, was chosen to describe the oligosaccharide synthesis, and one parameter set was determined for various initial lactose concentrations. Copyright 1999 John Wiley & Sons, Inc.

Enzymatic acylglycerol synthesis in a membrane bioreactor

Lipases can catalyze the esterification reaction in a two-phase system. TheCandida rugosa lipase-catalyzed esterification of decanoic acid with glycerol is described in this work for an emulsion system and for a hydrophilic membrane bioreactor. The enzymatic activity is studied in relation to the interface area between the two phases, the enzyme load and the reactor volume. The initial rate per unit interface area, the interfacial activity, is roughly equal for both systems indicating that the cellulose membrane does not hinder the esterification. Because the interfacial activities are equal, the volumetric activity of a membrane system is only specific area related, so a hollow fiber membrane device is preferable. The activity is also a function of the enzyme load. The optimum load in a hydrophilic membrane reactor is one to three times the amount of a monolayer, while in an emulsion system several times this amount. This could indicate that in the emulsion system the adsorption is in a dynamic state while at the membrane surface the adsorption reached its equilibrium state.

On-line water removal during enzymatic triacylglycerol synthesis by means of pervaporation

Abstract Triacylglycerols can be synthesized from glycerol and fatty acids. During this equilibrium reaction water is produced, therefore a mixture of mono-, di- and triesters is obtained. One way to produce an excess of triacylglycerols is to remove the water produced during synthesis. This can be realized in an immobilized enzyme pervaporation system. The enzyme is immobilized onto the lumen side of a cellulose membrane where the organic phase is present. Air circulates at the shell side and the water activity is controlled with the use of a condenser. The lipase catalyzed esterification of decanoic acid and partial glycerides is studied in this reactor. The system is reaction limited. Only at low water activity conditions, an excess of triacylglycerols is obtained. The enzyme activity at the start of the experiments is independent of the water activity within the range studied. Stability is influenced: After 600 hours the activity is 26% of the activity at the start at aw = 0.1 and 71% at aw = 0.45.

Enzymatic synthesis of monoglycerides in a membrane bioreactor with an in-line adsorption column

The chemical synthesis of monoglycerides requires high temperatures, which may lead to the polymerization of unsaturated fatty acids. The enzymatic synthesis of these esters is performed at moderate temperatures and, hence, polymerization is avoided. However, enzymatic processes often end up with a mixture of the product, by-product, substrate and enzyme. An alternative process is an immobilized enzyme membrane reactor equipped with an inline adsorption column to adsorb the monoglycerides, preferentially onto the adsorbate. A silica 60 column has shown preferential adsorption of monocaprinate. The adsorption of a mixture of decanoic acid, mono- and diglycerides is based on two different mechanisms. The decanoic acid will interact with hydroxyl groups at the silica gel surface, which results in a noncompetitive decanoic acid adsorption onto 25% of the silica gel surface. On the remaining part of the silica gel surface, mono- and diglycerides adsorb competitively. When a mild eluant is used, such as 5% ethanol in hexane, only the competitively adsorbed molecules are desorbed. This results in a purification factor of approximately 90% after desorption. The column can be desorbed off-line in a continuous membrane/repeated batch column process. This results in an estimated production of monoglycerides of 60 mol (15 kg) of monoester per gram enzyme.

Influence of membrane morphology on pore activation in membrane emulsification

Abstract The low throughput of the disperse phase is one of the issues in cross-flow membrane emulsification. This is apparent in the low percentage of pores at which droplets are formed (few active pores). To determine the effect of membrane morphology on pore activation, we developed and experimentally validated a model that describes the flow phenomena in and under a membrane with uniform pores (microsieve). In this model the membrane is divided into two parts: the toplayer and the membrane substructure. The model was validated with a larger-scale physical analogon. It predicts a linear increase of the number of active pores with increasing transmembrane pressure, while the pressure difference over the active pores is independent of the transmembrane pressure as long as not all pores are active. Although the resistance of the microsieve substructure was found to be four times lower than the resistance of a single pore, the resistance of the membrane substructure had a large effect on the activation of pores. Hence, the number of active pores can be increased by increasing the ratio of flow resistance in the pores and the flow resistance in the membrane substructure. Preliminary experiments show that the gradual increase in active pores at a ceramic membrane surface can be explained in the same way.

Membrane modification to avoid wettability changes due to protein adsorption in an emulsion/membrane bioreactor

This study addresses problems encountered with an emulsion/membrane bioreactor. In this reactor, enzyme- (lipase) catalyzed hydrolysis in an emulsion was combined with two in-line separation steps. One is carried out with a hydrophilic membrane, to separate the water phase, the other with a hydrophobic membrane, to separate the oil phase. In the absence of enzyme, sunflower oil/water emulsions with an oil fraction between 0.3 and 0.7 could be separated with both membranes operating simultaneously. However, two problems arose with emulsions containing lipase. First, the flux through both the hydrophilic and the hydrophobic membranes decreased with exposure to the enzyme. Second, the hydrophobic membrane showed a loss of selectivity demonstrated by permeation of both the oil phase and the water phase through the hydrophobic membrane at low transmembrane pressure. These phenomena can be explained by protein (i.e. lipase) adsorption to the polymer surface within the pores of the membrane. It was proven that lipase was present at the hydrophilic membrane and that this, in part, explains the flux decrease of the hydrophilic membrane. To prevent the observed loss of selectivity with exposure to protein, the hydrophobic polypropylene membrane (Enka) was modified with block copolymers of propylene oxide (PO) and ethylene oxide (EO). These block copolymers act as a steric hindrance for proteins that come near the surface. The modification was successful: After 10 days of continuous operation the minimum transmembrane pressure at which water could permeate through an F 108-modified membrane was 0.5 bar, the same value as that observed in the beginning of the experiment. This indicates that loss of selectivity due to protein adsorption is prevented by the modification of the membrane.

The nature of fatty acid modifies the equilibrium position in the esterification catalyzed by lipase.

The equilibrium position in lipase mediated esterification of various fatty acids and butanol was studied. The influence of the chain length and the presence of unsaturations in the fatty acids on the equilibrium position was measured and predicted. To predict equilibrium position the program TREP extended (TREPEX) based on the UNIFAC group contribution method was used. Using an equilibrium constant of 35, calculated on the basis of thermodynamic activities, the equilibrium position between butanol and saturated and/or unsaturated fatty acids with different chain lengths can be predicted. The ester mole fraction at equilibrium increases with the fatty acid chain length, and for fatty acids with the same carbon number, the highest values are found for unsaturated fatty acids. For reaction systems containing two saturated fatty acids, a slightly higher mole fraction is obtained for the fatty acid with the higher chain length, while for mixtures consisting of saturated and unsaturated fatty acids, the mole fractions of the unsaturated esters are lower than those of the saturated ones, regardless the chain length of the fatty acid. These experimental results are in good agreement with the calculations with TREPEX.

Lipase Mediated Resolution of γ-Branched Chain Fatty Acid Methyl Esters

Kinetic resolution of the branched chain fatty acid (BCFA) esters 4-methylhexanoic acid methyl ester (4) and 4-methyloctanoic acid methyl ester (5) was investigated using a series of hydrolases as catalysts. In the transesterification of these methyl esters to their butyl esters, two enzymes showed good conversion and a moderate enantiomeric ratio (E). In the transesterification of 4, an E of 2 was obtained for the reaction catalysed by Rhizomucor miehei lipase, whereas Candida antarctica lipase B (CALB) showed an E of 5. In the conversion of 5 to the butyl ester, Rhizomucor miehei lipase was unselective whereas CALB gave an E of 8. Apparently, changing from an ethyl group to a butyl group at the chiral centre leads to an improved chiral recognition by CALB. The lipases displayed complementary enantiomeric preference. Rhizomucor miehei lipase favours the S-enantiomer of 4 while CALB preferentially transforms the R-cnantiomer of both substrates. Molecular modelling studies supported the measured stereochem...

Multicomponent diffusion in dialysis membranes

Abstract Multicomponent diffusion through porous media is usually described by an effective diffusivity for each component. In such a diffusivity many different effects are lumped together, which makes its behaviour very difficult to understand. In this study we use a different approach in which each species has a driving force which is counteracted by friction due to its motion relative to the surroundings. The resulting equation is a difference form of what is known as the generalized Maxwell—Stefan equation (GMS). We apply this to describe transport through cellulose dialysis membranes. Friction between water and the membrane matrix is determined by isobaric dialysis experiments in mixtures with methanol, ethanol and 2-propanol. The water—membrane friction strongly depends on the water content. The friction of methanol or ethanol with the membrane is almost constant, while that of 2-propanol decreases with an increase in the 2-propanol concentration. The resulting friction coefficients give a quantitative description of transport of a ternary liquid mixture through the membrane. Using similar mixtures with a hollow fibre device shows that only the external area of the fibre bundle is effectively used. Apparently there is insufficient flow of the external phase between the fibres. In a second set of experiments a multicomponent system is studied. At the feed side of the membrane a solution of water, 2-propanol and sodium oleate is applied; on the permeate side a NaCl solution. A small pressure gradient from feed to permeate is applied. Initially a mass flux against the pressure gradient is observed. After some time the flux changes direction and becomes two to ten times larger than the permeation rate would be for the feed solution alone with the same applied pressure. These effects cannot be explained using effective diffusivities, but they can be understood qualitatively from the GMS equations.