On-line water removal during enzymatic asymmetric esterification in organic media

Summary Enantioselective esterification of 2-bromopropionic acid with n-butanol using Candida cylindracea lipase was carried out in n-pentane at various initial water contents. Reaction rate as well as enantioselectivity decreased at high water content. A heteroazeotropic distillation method was applicable to remove the excess water continuously and to work at the optimum reaction conditions.     

Real time measurement and control of thermodynamic water activities for enzymatic catalysis in hexane

The esterification reaction of geraniol with acetic acid catalyzed by immobilized Candida antarctica lipase B was studied in hexane using a pervaporation-assisted batch reactor. The effect of thermodynamic water activity (a(w)) on the initial reaction rate was investigated at a(w) ranging from 0.02 to 1.0. The a(w) was monitored on-line in real time. a(w) was actively controlled throughout the reaction by using highly water-selective membrane pervaporation. This novel combination of a(w) sensing and control eliminates changes in a(w) during the reaction even in the initial phase of relatively rapid water release during an esterification. No chemicals are introduced for a(w) control, and no purge gases or liquids are needed. A maximum in the initial reaction rate was found approximately at a(w)=0.1. The initial reaction rate declined quickly at higher a(w), and dropped precipitously at lower a(w).     

Pervaporation Aided Esterification of Carboxylic Acids with Ethanol Catalyzed by Porcine Pancreatic Lipase

The results of pervaporation-coupled esterification of various carboxylic acids with ethanol catalyzed by Porcine pancreatic lipase are reported. The effect of lipase and substrate concentrations has been studied and the advantage of pervaporation on the equilibrium conversion has been deduced. The kinetics of reaction were analyzed with a three-parameter model which coupled the effect of pervaporation. The intrinsic kinetic constants for all the reactions were estimated and correlated with the carbon number, an indicator of hydrophobicity of the acids. It was found that the rate constant increases with decrease in carbon number. The experimental concentration profiles were simulated from the model for all the reactions and the model prediction was found to be reasonably good. The water permeability was also correlated well with acid hydrophobicity. The pervaporation coupled reaction efficiency, as represented by the reaction time for equilibrium conversion, was found to bear a profound relation to membrane surface area per unit volume of the reaction mixture (A/V). The time for equilibrium conversion was found to decrease with an increase in A/V value, reaching a minimum and then increasing with a further increase of A/V. A probable explanation has been postulated for such an observation.     

Pervaporation Aided Esterification of Carboxylic Acids with Ethanol Catalyzed by Porcine pancreatic Lipase

The results of pervaporation-coupled esterification of various carboxylic acids with ethanol catalyzed by Porcine pancreatic lipase are reported. The effect of lipase and substrate concentrations has been studied and the advantage of pervaporation on the equilibrium conversion has been deduced. The kinetics of reaction were analyzed with a three-parameter model which coupled the effect of pervaporation. The intrinsic kinetic constants for all the reactions were estimated and correlated with the carbon number, an indicator of hydrophobicity of the acids. It was found that the rate constant increases with decrease in carbon number. The experimental concentration profiles were simulated from the model for all the reactions and the model prediction was found to be reasonably good. The water permeability was also correlated well with acid hydrophobicity. The pervaporation coupled reaction efficiency, as represented by the reaction time for equilibrium conversion, was found to bear a profound relation to membrane surface area per unit volume of the reaction mixture (A/V). The time for equilibrium conversion was found to decrease with an increase in A/V value, reaching a minimum and then increasing with a further increase of A/V. A probable explanation has been postulated for such an observation.     

The enzymatic synthesis of glucosylmyristate as a reaction model for general considerations on 'sugar esters' production

'Sugar esters' are non-ionic biodegradable surfactant which are potentially attractive for the cosmetic and food market. Unfortunately, the formation of by-products by chemical synthesis affects the quality of the surfactant. Enzymatic synthesis is a promising and environmentally friendly approach preventing this problem but requiring a proper setting up of the reaction system (membrane reactor, azeotropic mixture, pervaporation system, etc.) for the control of the water activity so that the enzyme efficiency is often negatively affected. In this paper, the synthesis of glucosylmyristate by Novozym 435 is taken as a model to illustrate the general features of the 'sugar esters' synthesis, with particularly regard to the influence on the synthesis of the pre-reaction treatment of the enzyme and reaction mixture, water adsorbents, reaction solvents, products and reagents. The aim is reached by placing the water adsorbents in contact with the enzyme preparation, so that high dehydration efficiency is achieved and the other factors affecting the kinetic of the synthesis are better highlighted.     

Lipase-catalyzed synthesis of geranyl acetate in n-hexane with membrane-mediated water removal.

The esterification of geraniol with acetic acid in n-hexane was investigated. A commercial lipase preparation from Candida antarctica was used as catalyst. The equilibrium conversion (no water removal) was found to be 94% for the reaction of 0.1 M alcohol and 0.1 M acid in n-hexane at 30 degrees C. This was shown by both hydrolysis and esterification reactions. The activation energy of reaction over the temperature range 10 degrees to 50 degrees C was found to be 16 kJ/mol. The standard heat of reaction was -28 kJ/mol. Membrane pervaporation using a cellulose acetate/ceramic composite membrane was then employed for selective removal of water from the reaction mixture. The membrane was highly effective at removing water while retaining all reaction components. Negligible transport of the solvent n-hexane was observed. Water removal by pervaporation increased the reaction rate by approximately 150% and increased steady-state conversion to 100%.     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

Pervaporative butanol fermentation by Clostridium acetobutylicum B18

Extractive acetone-butanol-ethanol (ABE) fermentation was carried out successfully using pervaporation and a low-acid-producing Clostridium acetobutylicum B18. A pervaporation module with 0.17 m(2) of surface area was made of silicone membrane of 240 mum thickness. Pervaporation experiments using make-up solutions showed that butanol and acetone fluxes increased linearly with their concentrations in the aqueous phase. Fickian diffusion coefficients were constants for fixed air flow rates, and increased at higher sweep air flow rates. During batch and fed-batch fermentations, pervaporation at an air flow rate of 8 L/min removed butanol and acetone efficiently. Butanol concentration was maintained below 4.5 g/L even though Clostridium acetobutylicum B18 produced butanol steadily. Pervaporation could not remove organic acids efficiently, but organic acids did not accumulate because strain B18 produced little organic acid and recycled added organic acids efficiently. With pervaporation, glucose consumption rate increased compared to without pervaporation, and up to 160 g/L of glucose was consumed during 80 h. Cell growth was not inhibited by possible salt accumulation or oxygen diffusion through the silicone tubing. The culture volume was maintained relatively constant during fed-batch operation because of an offsetting effect of water and product removal by pervaporation and addition of nutrient supplements. (c) 1994 John Wiley & Sons, Inc.     

Lipase reaction in AOT-isooctane reversed micelles: effect of water on equilibria

The effect of water on equilibria for hydrolytic reaction in reversed micelles has been investigated using lipase as a model enzyme. The effect of water on equilibria has been ignored for hydrolase reactions in an aqueous phase. In a reversed micellar system, however, the equilibrium of the lipase reaction was changed when water was added during the hydrolytic reaction. Furthermore, equilibrium fractional conversion is affected by the initial water concentration, being shifted to higher values with higher water concentrations, with other reaction conditions being held constant, indicating that the reaction should be regarded as a two-substrate process. Equations corresponding to a two-substrate, second-order reversible model are derived and used for further analysis. The progress curves predicted from the rate equations agree very well with the experimental results under various reaction conditions. The values of the molar ratio of water to surfactant (R) which maximize the initial reaction rate and maximum fractional conversion is predictable from the derived rate equations and the resulting relationship between R and the kinetic constants.     

Comparison of methods for thermolysin-catalyzed peptide synthesis including a novel more active catalyst

This is a comparative study of the performance of thermolysin for enzymatic peptide synthesis by reversed hydrolysis in several different reaction systems. Z-Gln-Leu-NH(2) was synthesized in acetonitrile containing 5% water (with various catalyst preparation methods) as well as by the "solid-to-solid" and frozen aqueous methods. Reaction rates (values in nanomoles per minute per milligram) in acetonitrile depended significantly on the method of addition of enzyme: (a) direct suspension in the reaction mixture as freeze-dried powders gave 60 to 95; (b) addition as an aqueous solution, so that enzyme precipitates on mixing with acetonitrile, gave 230; (c) addition as an aqueous suspension gave a remarkable increase in reaction rates (up to 780); (d) immobilized enzymes (adsorbed at saturating loading on celite, silica, Amberlite XAD-7, or polypropylene, then dried by propanol rinsing) all gave <230. It is postulated that, starting with the enzyme already in the form of solid particles in aqueous buffer, there is a minimum chance of alteration of its optimal conformation during transfer to the organic medium. For solid-to-solid synthesis with 10% water content we found initial rates of 670 under optimized conditions. In frozen aqueous synthesis, rates were <10. Equilibrium yields were always around 60% in low water organic solvent, whereas they were found to >80% in the aqueous systems studied.     

Pervaporation performance of a composite bacterial cellulose membrane: dehydration of binary aqueous–organic mixtures

Summary Acetobacter xylinum (Gluconacetobacter xylinus) is a bacterium that produces extracellular cellulose under static culture conditions. The highly reticulated cellulose matrix along with the entrapped cellulose-forming bacteria is commonly referred to as a pellicle. The processed bacterial cellulose membrane/film was modified into a composite bacterial cellulose membrane (CBCM) for pervaporation separation of aqueous–organic mixtures. The CBCM was prepared by coating with alginate or alginate+polyvinylpyrrolidone and cross-linking with glutaraldehyde. The pervaporation performance was determined using aqueous–organic mixtures such as, 1:1 (v/v) water–ethanol, water–isopropanol and water–acetone. The pervaporation performance of the CBCM was more effective for zeotropic mixtures (water–acetone) in comparison to the investigated azeotropic mixtures (water–ethanol and water–isopropanol). The selectivity of CBCM was found to be 4.8, 8.8, 19.8 for water–ethanol, water–isopropanol and water–acetone mixtures, respectively. The permeation flux for the water–acetone mixture was found to be 235 ml/m²/h. The present investigation demonstrated that the CBCM could be employed to concentrate azeotropic as well as zeotrope forming binary mixtures by preferential pervaporation of water, with low energy requirements in contrast to the established method of distillation. In addition, the effects of feed composition, operating temperature, membrane thickness, and method of CBCM preparation on pervaporation performance have been evaluated. Investigations with the CBCM revealed that 94.5% ethanol, 98% acetone and 98.5% isopropanol concentrations could be attained from the initial 50% aqueous mixtures of these chemicals by way of pervaporation. In the case of the isopropanol–water mixture the resolving property of the membrane was more evident as the concentration arrived at was 98.5%, in contrast to other binary mixtures. The surface characteristics of the CBCM were revealed by scanning electron microscopy. In view of its properties the CBCM can be useful for pervaporation separation of these chemicals at moderate temperatures and pressure. The CBCM could be employed in the downstream processing of heat-labile and flavor-imparting volatile molecules in the field of food biotechnology and fabrication of membrane bioreactors for on-line product purification. Further studies are under progress to use the membrane for the immobilization of food processing enzymes.     

Increased productivity of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> fermentation of acetone,butanol, and ethanol by pervaporation through supported ionic liquid membrane

Pervaporation proved to be one of the best methods to remove solvents out of a solvent producing Clostridium acetobutylicum culture. By using an ionic liquid (IL)-polydimethylsiloxane (PDMS) ultrafiltration membrane (pore size 60 nm), we could guarantee high stability and selectivity during all measurements carried out at 37°C. Overall solvent productivity of fermentation connected with continuous product removal by pervaporation was 2.34 g l⁻¹ h⁻¹. The supported ionic liquid membrane (SILM) was impregnated with 15 wt% of a novel ionic liquid (tetrapropylammonium tetracyano-borate) and 85 wt% of polydimethylsiloxane. Pervaporation, accomplished with the optimized SILM, led to stable and efficient removal of the solvents butan-1-ol and acetone out of a C. acetobutylicum culture. By pervaporation through SILM, we removed more butan-1-ol than C. acetobutylicum was able to produce. Therefore, we added an extra dose of butan-1-ol to run fermentation on limiting values where the bacteria would still be able to survive its lethal concentration (15.82 g/l). After pervaporation was switched off, the bacteria died from high concentration of butan-1-ol, which they produced.     

Intramolecular esterification by lipase powder in microaqueous benzene: effect of moisture content

In view of the biochemical reaction catalyzed by enzyme powder suspended in a water-insoluble organic solvent, an equation was derived to estimate the amount of water bound to the enzyme powder. With this equation, an apparent adsorption isotherm between free water (water freely dissolved in benzene) and bound water (water bound to crude lipase powder of Pseudomonas fluorescens) was obtained. A direct lactonization reaction (synthesis of cyclopentadenolide from 15-hydroxypen-tadecanoic acid) catalyzed by crude lipase powder of Pseudomonas fluorescens was carried out batchwise in microaqueous benzene at 40oC. A kinetic model of the enzymatic reversible lactonization reaction was derived, from which the effect of moisture content on the initial reaction rate with a fully hydrated enzyme was mathematically expressed. The observed initial reaction rate first increased, then decreased with increasing moisture content, giving rise to the maximum rate at a certain level of the moisture content. The drop in the reaction rate at lower moisture content was due to a lesser hydration of the enzyme molecule (hydration-limited) and the decrease in the reaction rate at higher moisture content was attributed to the dependence of the true initial rate of the reversible reaction on the moisture content (true reversible reaction limited), and could be simulated by the kinetic model. The equilibrium yield approached 100% at a lower moisture content.     

Lipase-Catalyzed Reactions in Reverse Micelles Formed by Soybean Lecithin

Reverse micelles formed by soybean lecithin in isooctane were used as a reaction medium for both the lipase-catalyzed hydrolysis as well as the synthesis of lipids. Neither reaction appears to follow Michaelis-Menten kinetics and it is suggested that the rates are diffusion controlled. The hydrolysis of para-nitrophenylpalmitate (PNPP) and, in particular, the pH-dependency of the lipase-catalyzed hydrolysis was then examined. The highest rate of reaction occurred at pH_opt = 5–5.5, which was the same in water and lecithin reverse micelles, as well as in reverse micelles formed by bis(2-ethylhexyl)-sulfosuccinate (AOT) in isooctane. The dependence of the reaction rate on the water content of the micellar system was investigated for the same reaction. The maximal rate was found at an extremely low water content, i.e. at W_o = 2.2 (W_o = [H₂O]/[Lecithin]). The temperature stability of the lipase in lecithin reverse micelles was also studied and found to be greater than in aqueous solutions. Studies of the dependence of the relative initial velocity on temperature have shown that the highest rate in reverse micelles is obtained at 60d`C.     

Pervaporation-aided enzymatic esterifications in non-conventional media

This paper focuses on enzymatic esterifications in non-conventional media (organic solvents, ionic liquids, and solvent-free systems) with reference to the water removal. Different types of water removal techniques are reviewed with a special emphasis on pervaporation. Pervaporation is a separation process in which liquid is transported through a selective membrane with simultaneous evaporation of permeates. In an integrated process where pervaporation is coupled with a bioreactor where esterification is performed, selective removal of water or other esterification products can be achieved. In this manner benefit can be doubled, due to the equilibrium shift and possible pure product recovery. Available literature on esterifications coupled with pervaporation is presented in detail. Reviewed examples are divided according to the type of reaction media.     

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.     

Enzymatic synthesis of butyl hydroxycinnamates and their inhibitory effects on LDL-oxidation

The potential of the Aspergillus niger type A feruloyl esterase (AnFaeA) for the synthesis of various phenolic acid esters was examined using a ternary-organic reaction system consisting of a mixture of n-hexane, 1- or 2-butanol and water. Reaction parameters including the type of methyl hydroxycinnamate, the composition of the reaction media, the temperature, and the substrate concentration were investigated to evaluate their effect on initial rate and conversion to butyl esters of sinapic acids. Optimisation of the reaction parameters lead to 78% and 9% yield for the synthesis of 1-butyl and 2-butyl sinapate, respectively. For the first time, a feruloyl esterase was introduced in the reaction system as cross-linked enzyme aggregates (CLEAs), after optimisation of the immobilisation procedure, allowing the recycling and reuse of the biocatalyst. The inhibition of copper-induced LDL oxidation by hydroxycinnamic acids and their corresponding butyl esters was investigated in vitro. Kinetic analysis of the antioxidation process demonstrates that sinapate derivatives are effective antioxidants indicating that esterification increases the free acid's antioxidant activity especially on dimethoxylated compounds such as sinapic acid compared to methoxy-hydroxy-compounds such as ferulic acid.     

Lipase-Catalyzed Synthesis and Hydrolysis of Cholesterol Oleate in Aot/Isooctane Microemulsions

We have examined a lipase-catalyzed bidirectional ester synthesis/hydrolysis reaction in a water-in-oil microemulsion system. The reactants were cholesterol (alcohol), oleic acid (acid) and cholesterol oleate (ester), and the solvent system consisted of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/water. The reactions were assayed by using [³H]oleic acid, [³H]cholesterol, or [³H]cholesterol oleate for the synthesis and hydrolysis reactions, respectively (separate incubations). The lipase that we used derived from Candida cylindracea, and was used at a concentration of 0.1mg/ml microemulsion. The reactions were performed at 22°C as the reactions proceeded more slowly at higher temperatures. With the initial reactant concentrations set to 10 mM cholesterol, 1 min oleic acid, and 1 mM cholesterol oleate, it was observed that the optimal [H₂O]/[AOT] ratio was at about 9 both for the esterification reaction and for the hydrolysis reaction (after 24 h). The hydrolysis reaction was slower than the synthesis reaction at all [H₂O]/[AOT] ratios studied (0-20), but the difference in reaction yield for the synthesis and the hydrolysis reactions became smaller as the reaction time increased (up to 11 days). When the reaction yield was followed as a time function, it was observed that about 80% of the oleic acid was esterified within 3 days of reaction ([H₂O]/[AOT] ratio of 6), whereas the corresponding value of 80% hydrolysis of cholesterol oleate was reached within 11 days. The results of the present study indicate that by choosing optimal reactant concentrations and reaction conditions, it is at least in part possible to determine the direction of the lipase-catalyzed synthesis/hydrolysis reaction.     

Kinetics of enzymatic solid-to-solid peptide synthesis: synthesis of Z-aspartame and control of acid-base conditions by using inorganic salts

Enzymatic peptide synthesis can be carried out efficiently in solid-to-solid reaction mixtures with 10% (w/w) water added to a mixture of substrates. The final reaction mass contains >/=80% (by weight) of product. This article deals with acid-base effects in such reaction mixtures and the consequences for the enzyme. In the Thermoase-catalyzed synthesis of Z-Asp-Phe-OMe, the reaction rate is strongly dependent on the amount of basic salts added to the system. The rate increases 20 times, as the KHCO(3) or K(2)CO(3) added is raised 2.25-fold from an amount equimolar to the Phe-OMe. HCL starting material. With further increases in KHCO(3) addition, the initial rate remains at the maximum, but with K(2)CO(3) it drops sharply. Addition of NaHCO(3) is less effective, but rates are faster if more water is used. With >1.5 equivalents of basic salt, the final yield of the reaction decreases. Similar effects are observed when thermolysin catalyzes the same reaction, or Z-Gln-Leu-NH(2) synthesis. These effects can be rationalized using a model estimating the pH of these systems, taking into account the possible formation of up to ten different solid phases.     

Dehydration of an ethanol/water azeotrope by novel organic-inorganic hybrid membranes based on quaternized chitosan and tetraethoxysilane

To control swelling of quaternized chitosan (q-Chito) membranes, mixtures of q-Chito as an organic component and tetraethoxysilane (TEOS) as an inorganic component were prepared using the sol-gel reaction, and novel q-Chito/TEOS hybrid membranes were formed. In the separation of an ethanol/water azeotrope by pervaporation, the effect of TEOS content on the water/ethanol selectivity of q-Chito/TEOS hybrid membranes was investigated. Hybrid membranes containing up to 45 mol % TEOS exhibited higher water/ethanol selectivity than the q-Chito membrane. This resulted from depressed swelling of the membranes by formation of a cross-linked structure. However, introduction of excess TEOS led to greater swelling of the hybrid membranes. Therefore, the water/ethanol selectivity of the hybrid membranes containing more than 45 mol % TEOS was lower than that of the q-Chito membrane. The relationship between the structure of q-Chito/TEOS hybrid membranes and their permeation and separation characteristics during pervaporation of an ethanol/water azeotrope is discussed in detail.