Optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phase system containing ionic liquids and t-BuOH

Selective lipase-catalyzed synthesis of glucose fatty acid esters in two-phase systems consisting of an ionic liquid (1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF₄] or 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIM][PF₆]) and t-butanol as organic solvent was investigated. The best enzyme was commercially available lipase B from Candida antarctica (CAL-B), but also lipase from Thermomyces lanuginosa (TLL) gave good conversion. After thorough optimization of several reaction conditions (chain-length and type of acyl donor, temperature, reaction time, percentage of co-solvent) conversions up to 60% could be achieved using fatty acid vinyl ester as acyl donors in [BMIM][PF₆] in the presence of 40% t-BuOH with CAL-B at 60 °C.     

Optimization of lipase-catalyzed glucose ester synthesis in ionic liquids

Lipase-catalyzed esterification of glucose with fatty acids in ionic liquids (ILs) mixture was investigated by using supersaturated glucose solution. The effect of ILs mixture ratio, substrate ratio, lipase content, and temperature on the activity and stability of lipase was also studied. The highest yield of sugar ester was obtained in a mixture of 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim][TfO]) and 1-methyl-3-octylimidazolium bis[(trifluoromethyl)-sulfonyl]amide ([Omim][Tf₂N]) with a volume ratio of 9:1, while Novozym 435 (Candida antarctica type B lipase immobilized on acrylic resin) showed the optimal stability and activity in a mixture of [Bmim][TfO] and [Omim][Tf₂N] with a 1:1 volume ratio. Reuse of lipase and ILs was successfully carried out at the optimized reaction conditions. After 5 times reuse of Novozym 435 and ILs, 78% of initial activity was remained.     

Competitive lipase-catalyzed ester hydrolysis and ammoniolysis in organic solvents; equilibrium model of a solid-liquid-vapor system.

Enzymatic ester hydrolysis and ammoniolysis were performed as competitive reactions in methyl isobutyl ketone without a separate aqueous phase. The reaction system contained solid ammonium bicarbonate, which dissolved as water, ammonia, and carbon dioxide. During the reaction an organic liquid phase, a vapor phase, and at least one solid phase are present. The overall equilibrium composition of this multiphase system is a complex function of the reaction equilibria and several phase equilibria. To gain a quantitative understanding of this system a mathematical model was developed and evaluated. The model is based on the mass balances for a closed batch system and straightforward relations for the reaction equilibria and the solubility equilibria of ammonium bicarbonate, the fatty acid ammonium salt, water, ammonia, and carbon dioxide. For butyl butyrate as a model ester and Candida antarctica lipase B as the biocatalyst this equilibrium model describes the experiments satisfactorily. The model predicts that high equilibrium yields of butyric acid can be achieved only in the absence of ammoniolysis or in the presence of a separate water phase. However, high yields of butyramide should be possible if the water concentration is fixed at a low level and a more suited source of ammonia is applied.     

Study of vitamin ester synthesis by lipase-catalyzed transesterification in organic media

Immobilized lipase from Candida antarctica (Novozym 435) was used in organic media to catalyze esterifications of vitamins (ascorbic acid and retinol) from hydroxy acid. We described the synthesis of retinyl L-lactate by transesterification between retinol and L-methyl lactate with yield reaching 90% and the synthesis of ascorbyl L-lactate by transesterification between ascorbic acid and L-methyl lactate with yield reaching 80%. The kinetic study of the esterification of vitamins with L-methyl lactate in organic media has been carried out and agrees with ping-pong-ordered Bi-Bi when the initial vitamin concentration is low. When initial vitamin concentration is high, the kinetic is similar to a hybrid ping-pong-ordered Bi Bi or hybrid ping-pong-random Bi Bi mechanism. However, with high initial substrate concentration, change of the kinetic by other phenomena, such as interaction of substrates with molecular sieves, adsorption of the methanol formed, and decreases of substrate diffusion, could be considered. It is obvious that in these conditions, classical enzymology (i.e., Michaelian enzymology) cannot be used for the interpretation of results.     

Optimized lipase-catalyzed synthesis of adipate ester in a solvent-free system

Immobilized Candida antarctica lipase-catalyzed esterification of adipic acid and oleyl alcohol was investigated in a solvent-free system (SFS). Optimum conditions for adipate ester synthesis in a stirred-tank reactor were determined by the response surface methodology (RSM) approach with respect to important reaction parameters including time, temperature, agitation speed, and amount of enzyme. A high conversion yield was achieved using low enzyme amounts of 2.5% w/w at 60°C, reaction time of 438 min, and agitation speed of 500 rpm. The good correlation between predicted value (96.0%) and actual value (95.5%) implies that the model derived from RSM allows better understanding of the effect of important reaction parameters on the lipase-catalyzed synthesis of adipate ester in an organic solvent-free system. Higher volumetric productivity compared to a solvent-based system was also offered by SFS. The results demonstrate that the solvent-free system is efficient for enzymatic synthesis of adipate ester.     

Thermodynamic and kinetic parameters of lipase-catalyzed ester hydrolysis in biphasic systems with varying organic solvents

Kinetics of lipase-catalyzed hydrolysis of esters were modeled using reactant activities for aqueous-organic, biphasic systems. By using thermodynamic activities of the substrates in ordinary rate equations, the kinetic parameters were corrected for the contribution of substrate-solvent interactions and a uniform quantification of the substrates for lipase attached to the interface can be achieved. The kinetic parameters, on the basis of their thermodynamic activities, should be constant in different systems, provided that the solvents do not interfere with the binding of the substrates to the enzyme nor affect the catalytic mechanism. Experimental and computational methods on how to obtain the thermodynamic activities of the substrates are presented. Initial rates were determined for Pseudomonas cepacia lipase (PcL)-catalyzed hydrolysis of decyl chloroacetate in dynamic emulsions with various solvents. The thermodynamic equilibrium and corrected kinetic constants for this reaction appeared to be similar in various systems. The kinetics of PcL in an isooctane-aqueous biphasic system could be adequately described with the rate equation for a ping-pong mechanism. The observed inhibitory effect of decanol appeared to be a consequence of this mechanism, allowing the backreaction of the decanol with the chloroacetyl-enzyme complex. The kinetic performance of PcL in systems with toluene, dibutyl ether, and methyl isobutyl ketone could be less well described. The possible causes for this and for the remaining differences in corrected kinetic parameters are discussed. (c) 1995 John Wiley & Sons, Inc.     

Lipase catalysed synthesis of sugar ester in organic solvents

Summary The synthesis of sugar esters catalysed by lipase in organic solvents was studied. Immobilized Candida and Mucor miehei lipase catalysed the synthesis of fructose and glucose esters of stearic acid in tertiary butyl alcohol with yields of 10 to 24 %. In the presence of phenyl or butyl boronic acid synthesis of glucose ester was achieved in hexane, heptane, benzene and toluene. The only positive reaction on disaccharides was found with palatinose.     

Optimization of lipase-catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids by response surface methodology

Lipase-catalyzed caffeic acid phenethyl ester (CAPE) synthesis in ionic liquid, 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf₂N]), was investigated in this study. The effects of several reaction conditions, including reaction time, reaction temperature, substrate molar ratio of phenethyl alcohol to caffeic acid (CA), and weight ratio of enzyme to CA, on CAPE yield were examined. In a single parameter study, the highest CAPE yield in [Emim][Tf₂N] was obtained at 70 °C with a substrate molar ratio of 30:1 and weight ratio of enzyme to CA of 15:1. Based on these results, response surface methodology (RSM) with a 3-level-4-factor central composite rotatable design (CCRD) was adopted to evaluate enzymatic synthesis of CAPE in [Emim][Tf₂N]. The four major factors were reaction time (36–60 h), reaction temperature (65–75 °C), substrate molar ratio of phenethyl alcohol to CA (20:1–40:1), and weight ratio of enzyme to CA (10:1–20:1). A quadratic equation model was used to analyze the experimental data at a 95 % confidence level (p < 0.05). A maximum conversion yield of 99.8 % was obtained under the optimized reaction conditions [60 h, 73.7 °C, substrate molar ratio of phenethyl alcohol to CA (27.1:1), and weight ratio of enzyme to CA (17.8:1)] established by our statistical method, whereas the experimental conversion yield was 96.6 ± 2 %.     

A comparison of lipase-catalyzed ester hydrolysis in reverse micelles, organic solvents, and biphasic systems

The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the "anhydrous" enzyme suspension approach. (c) 1995 John Wiley & Sons, Inc.     

Peptide and ester synthesis in organic solvents catalyzed by seryl proteases linked to alumina

Trypsin and alpha-chymotrypsin were immobilized to alumina-phosphocolamine complex, activated by glutaraldehyde. The immobilized enzymes show a great stability toward organic solvents miscible or immiscible with water. In the presence of a low concentration of water, the immobilized enzymes catalyzed transesterification reactions as well as peptide synthesis. The synthesized peptides were stable toward the immobilized enzymes.     

Selective lipase-catalyzed preparation of diol monobenzoates by transesterification and alcoholysis reactions in organic solvents

Lipases from Mucor miehei (MML) and Candida antarctica (CAL) are able to catalyze the monobenzoylation of the primary hydroxy group of 1,2- 1,4- or 1,5-diols with vinyl benzoate in an organic solvent, the reaction proceeding with high regioselectivity and moderate enantioselectivity. The lipase-catalyzed debenzoylation of 1,2-propanediol dibenzoate by alcoholysis with 1-octanol most satisfactorily occurred with Pseudomonas cepacia lipase absorbed onto celite that allowed also to prepare (R)-1-benzoyloxy-2-methylpropan-3-ol from 2-methyl-1,3-propanediol dibenzoate, a result complementary to MML-catalyzed benzoylation of 2-methyl-1,3-propanediol that affords the (S)-monobenzoate.     

Recovery of steroidal glucosiduronic acids from organic solvents containing anionic liquid ion-exchangers

Solutions of anionic liquid ion-exchangers in organic solvents are potentially useful for extracting steroidal glucosiduronic acids from biological fluids and for purifying mixtures of these acids by chromatography. If a glucosiduronic acid is to be isolated in pure form after either of these procedures, it is necessary to separate it from the ion-exchanger. Separation from an organic solution of tetraheptylammonium chloride may be accomplished by extraction with water under the following conditions, which promote transfer of a glucosiduronate to the aqueous phase: (1) an appropriate solvent (diluent) as the organic phase, (2) the presence in the two-phase mixture of an anion such as myristate or dodecyl sulphate to combine with the tetraheptylammonium ion, and (3) an increase of the pH of the aqueous phase in association with the presence of myristate or dodecyl sulphate. The foregoing factors apply also to removal of glucosiduronates from organic solutions of ion exchangers that are hydrochlorides of tertiary, secondary, or primary amines. Since these amines exert progressively less solubilizing effect for glucosiduronates as the pH of the aqueous phase is increased, the conjugates can be released from the organic phase by adjusting the pH to 10 and omitting the myristate or dodecyl sulphate.     

Thermodynamics of the lipase-catalyzed esterification of glycerol and n-octanoic acid in organic solvents and in the neat reaction mixture

The thermodynamics of the lipase-catalyzed esterification of glycerol with n-octanoic acid have been investigated with acetonitrile, benzene, and toluene as solvents and in the neat reaction mixture (no organic solvent added). This esterification reaction leads to five products: 1-monooctanoyl glycerol, 2-monooctanoyl glycerol, 1,2-dioctanoyl glycerol, 1,3-dioctanoyl glycerol and 1,2,3-trioctanoyl glycerol. This, in turn leads to a total of 12 reactions. Values of the equilibrium constants for these reactions have been measured (HPLC, GC, and LC/MS) at 37°C in the above mentioned media. The equilibrium constants range from 0.9 to 20.7, 0.20 to 8.0, 0.23 to 10.0, and 0.57 to 2.2 in acetonitrile, benzene, toluene, and neat media, respectively. Relative standard molar Gibbs free energies of formation Δ_fG_m⁰ of 1-monooctanoyl glycerol, 2-monooctanoyl glycerol, 1,2-dioctanoyl glycerol, 1,3-dioctanoyl glycerol and 1,2,3-trioctanoyl glycerol in the organic solvents and in the neat reaction mixture have been calculated and used to compactly summarize the thermodynamics of these reactions. The results show an approximate correlation with the permittivities of the solvents.     

A new aqueous biphasic system containing polypropylene glycol and a water-miscible ionic liquid

In this work, we proposed a novel aqueous biphasic system (ABS) composed of polypropylene glycol P400 (PPG P400) and hydrophilic ionic liquids (IL), 1-alkyl-3-methylimidazolium bromide (alkyl = ethyl or butyl), forming an upper polymer-rich phase and a lower IL-rich phase at ambient temperature. This new ABS can present interesting characteristics shared by ILs and polymers such as low volatility, good solvation ability, tunable physical properties, and high design capacity for achieving task-specific phase components to enhance the partitioning of target species. Ternary phase diagram of the novel ABS formed by PPG 400 and [C(2) mim]Br in water was measured at T = 298.15 K. Factors affecting the binodal curves such as the cation side alkyl chain length and the temperature were also evaluated. The results were successfully interpreted in terms of the kosmotropic/chaotropic nature of ILs. Furthermore, the phase behavior of the PPG-[C(2) mim]Br ABS is described by the NRTL model. Finally, the extraction potential of the proposed ABS was evaluated through its application to the extraction of the essential amino acids such as L-tryptophan and L-tyrosine. The partition coefficients here obtained demonstrated the fine potential of the proposed ABS for biomolecules separation.     

Lipase-catalyzed synthesis of glucose fatty acid ester using ionic liquids mixtures

Novozym 435-catalyzed synthesis of 6-O-lauroyl-d-glucose in ionic liquids (ILs) was investigated. The highest lipase activity was obtained in water-miscible [Bmim][TfO] which can dissolve high concentration of glucose, while the highest stability of lipase was shown in hydrophobic [Bmim][Tf(2)N]. The optimal activity and stability of lipase could be obtained in [Bmim][TfO] and [Bmim][Tf(2)N] mixture (1:1, v/v). Specifically, the activity of lipase was increased from 1.1 to 2.9 micromolmin(-1)g(-1) by using supersaturated glucose solution in this mixture, compared with reaction using saturated solution. After 5 times reuse of lipase, 86% of initial activity was remained in this mixture, while the residual activity in pure [Bmim][TfO] was 36%. Therefore, the productivity obtained by using ILs mixtures was higher than those in pure ILs.     

Preparation of monomeric acrylic ester intermediates using lipase catalysed transesterifications in organic solvents

Summary Acrylate monoesters were synthesized on a preparative scale by the regioselective enzymatic transesterification of a range of diols dissolved in ethyl acrylate using a commercial lipase fromChromobacterium viscosum.     

Improving lipase-catalyzed enantioselective ammonolysis of phenylglycine methyl ester in organic solvent by in situ racemization

Lipase-catalyzed enantioselective ammonolysis of phenylglycine methyl ester was processed by in situ racemization with ammonium carbamate as the acyl acceptor. Using 1 mM benzaldehyde or 0.6 mM chloropyridoxal as the racemizing catalyst, 80% substrate conversion with an enantiomeric excess of the product of 95% were achieved at 20 °C after 7 h reaction.     

Separation of lipid-free egg yolk proteins by high-pressure liquid chromatography using solvents containing formic acid

A method for obtaining total protein patterns from lipid-containing systems, in particular egg yolk, is described. After dispersion of the yolk in 8 M guanidine hydrochloride solution, lipid is removed by extraction with chloroform-methanol and petrol. The protein solution is applied to a high-pressure liquid chromatograph and eluted with a gradient of formic acid, isopropanol, and acetonitrile. In measurements on a known yolk protein, duck apovitellenin I, the method did not cause irreversible formylation of N-terminal or other residues. The method was used (1) to compare protein patterns of whole yolk from hen and quail eggs; (2) to isolate and partially sequence quail apovitellenin I; and (3) to compare protein patterns of "white yolk" and "yellow yolk."     

Synthesis of chiral building blocks for organic synthesis via lipase-catalyzed reaction: New method of enhancing enzymatic reaction enantioselectivity

The optically active building blocks for organic synthesis: tertiary carbinols, antitumor lignan, liquid crystals, 1,3-diene and biscyclopropyl compounds were synthesized through lipase-catalyzed reaction. This paper discusses ways in which organic chemists can expand the applicability of lipase-catalyzed reactions for use in designing a synthetic strategy. Several excellent examples are described in which lipase-catalyzed reactions were involved as the key steps. Because lipase-catalyzed reactions often offer insufficient enantioselectivity, a new method to enhance the enantioselectivity of a lipase-catalyzed reaction was demonstrated. Thiacrown ether technology was typically used to synthesize new optically active ,-difluoro-γ-lactone.