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Optimization of 1-glyceryl benzoate production by enzymatic transesterification in organic solvents
Enzyme and Microbial Technology 46 (2010) 107–112
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Optimization of 1-glyceryl benzoate production by enzymatic transesterification in organic solvents Giovana Ceni a,b , Lindomar A. Lerin a , Juliana Faccin de Conto b , Cristiane Vendrusculo Brancher b , Patrícia Costa da Silva b , Geciane Toniazzo b , Helen Treichel b,∗ , Débora de Oliveira b , J. Vladimir Oliveira b , Enrique Guillermo Oestreicher a , Octavio Augusto Ceva Antunes a a
Universidade Federal do Rio de Janeiro, Instituto de Química, Cidade Universitária, 21941-909, Rio de Janeiro, RJ, Brazil Universidade Regional Integrada do Alto Uruguai e das Missões (URI), Campus de Erechim Depto de Engenharia de Alimentos, Av. 7 de Setembro, 1621, 99700-000, Erechim, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 19 May 2009 Received in revised form 8 July 2009 Accepted 22 September 2009 Keywords: Kinetics Transesterification 1-Glyceryl benzoate
a b s t r a c t This work is focused on the evaluation of reaction parameters involved in the enzymatic production of 1-glyceryl benzoate, an intermediate substance to carvedilol and propranolol synthesis, catalyzed by Candida antarctica lipase in different organic solvents. To our knowledge, no related study on this subject is available in the open literature. The main goals of the present investigation were to elucidate the relationship between relevant reaction variables and also to determine the optimum conditions for 1-glyceryl benzoate production. Results showed that the strategy adopted for the experimental design proved to be useful in maximizing the reaction conversion in 2-propanol as solvent with Novozym 435 as catalyst. The optimum conditions were found to be methyl benzoate to glycerol molar ratio of 1:1, stirring rate of 150 rpm, 50 ◦ C, enzyme concentration of 10 wt% at 36 h of reaction, with a resulting conversion to 1-glyceryl benzoate of about 29%. Reaction kinetics of 1-glyceryl benzoate production demonstrated that very satisfactory conversions (∼40%) were achieved after 70 h of reaction. © 2009 Elsevier Inc. All rights reserved.
1. Introduction The use and preparation of chiral pharmaceutical principles as single enantiomers is one of the most relevant goals in pharmaceutical science. Optically active ␣-monoacyl glycerol is a useful starting material for the preparation of chiral drugs, such as blockers [1]. For chiral drugs, opposite enantioforms act with different biological properties and the distomer could give undesirable effects [2]. The resolution of racemic compounds seems to be a valuable method for obtaining chiral compounds at high optical purity [3,4] through both enzymatic [3] and non-enzymatic [4] catalysts with a wide variety of substrates. The great disadvantage of standard kinetic resolution procedure is that a maximum yield of 50% of the desired product is obtained from the starting racemic material. In this sense, enzymes have been extensively investigated for asymmetric syntheses due to the growing demand for enantiopure intermediates and drugs [3]. It is often necessary to conduct these enzymatic reactions in organic solvents towards improving substrate solubility and shifting the reaction equilibrium to products formation.
∗ Corresponding author. E-mail address: [email protected] (H. Treichel). 0141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2009.09.011
It is well known that lipases (E.C. 3.1.1.3) are very suitable enzymes for organic synthesis because they accept a wide range of non-natural substrates, are stable and active in organic solvents, do not require cofactors, and are readily available from several microorganisms. In organic solvents, lipases can be more enantioselective and the solubility of hydrophobic substrates can be enhanced [5]. Possible approaches to the enzymatic resolution of chiral products include the enantioselective hydrolysis, transesterification or the direct esterification in non-aqueous medium using microbial lipases. In this sense, lipases are today the natural choice of biocatalysts as they show unique chemo-, regio- and enatioselectivities, which enable the production of novel drugs, agrochemicals and fine chemicals [6,7]. Selection of a lipase for a given application should be based on its substrate specificity such as fatty acid, alcohol, position (regio-) and stereospecificity, as well as temperature and pH stability. Related to the huge amount and variety of applications of lipases, expected to rise in the near future, the search for lipases with characteristic substrate specificity and stability at high temperatures and in organic solvents is of great interest [8]. Because of the low solubility of many esters in some alcohols, the substrates are typically dissolved in an organic solvent, such as n-heptane. This is especially true when dealing with nonpolar organic substances and several other polar compounds such as glycerol.
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Lipases were envisaged as biocatalysts for the transesterification of methyl benzoate with glycerol to yield 1-glyceryl benzoate as main product. This compound was the obvious choice to replace epichloridrin as a green intermediate for beta-blockers production typified by carvedilol and propranolol [9]. The antagonists of -adrenergic receptors (-blockers), as propranolol [10] and carvedilol, are important active pharmaceutical ingredients (APIs) used in the treatment of diseases as hypertension, cardiopathies, some kinds of hyh and glaucoma. This class of substances has received considerable attention recently from the pharmacological and chemical point of view. In spite of the importance of producing this intermediate (1glyceryl benzoate) no experimental data on this subject was found in the current scientific literature but one paper that makes use of vinyl benzoate a much more expensive substrate [1]. It may be opportune to call attention at this point to the fact that the establishment of world-wide biodiesel government programs, with the expected huge glycerol surplus, has prompted many attempts to produce high-value added products through glycerol transformation by enzyme-catalyzed reactions. Based on these aspects, the main objective of this work was to investigate the enzymatic production of 1-glyceryl benzoate in organic solvents. Preliminary, the solubility of substrates in different organic solvents and the stability of the commercial immobilized lipase (Novozym 435) in such solvents were evaluated. From the results obtained in this step, the optimization of the process conversion and a kinetic study of enzymatic transesterification of glycerol and methyl benzoate were carried out. 2. Materials and methods 2.1. Material Glycerol and methyl benzoate (Vetec, both 99.5% purity) were used as substrates for the transesterification reactions. The commercial immobilized lipase used in this work was Candida antarctica (Novozym 435) immobilized on a macroporous anionic resin, kindly supplied by Novozymes Brazil (Araucária, PR, Brazil). The organic solvents used in the experimental tests were acetone, 2-propanol, 2-methylbutanol, tert-butanol, n-hexane, n-heptane, isooctane, di-isopropylether, petroleum ether, tetrahydrofuran and n-decane (Quimex, 99.5% purity). For some experimental conditions, sodium (bis-2-ethyl-hexyl) sulfosuccinate (aerosol-OT or AOT) was used as surfactant to allow the formation of reverse micelles. 2.2. Lipase esterification activity The enzyme activity was determined as the initial rates in esterification reactions between lauric acid and n-propanol at a molar ratio of 3:1, temperature of 60 ◦ C and enzyme concentration of 5 wt% in relation to the substrates. At the beginning of the reaction, samples containing the mixture of lauric acid and n-propanol were collected and the lauric acid content was determined by titration with NaOH 0.04N. After the addition of the enzyme to the substrates, the mixture was kept at 60 ◦ C for
Table 1 Parameters of solvents and substrates used in the solubility study [11]. Solvent
Molar volume (cm3 /g mol)
Solubility parameter (MPa)1/2
Acetone n-Hexane n-Heptane Isooctane Tetrahydrofuran Di-isopropylether 2-Propanol n-Decane 2-Methylbutanol Petroleum ether Tert-butanol
74.0 131.6 147.4 166.1 81.7 142.1 76.8 196.0 109.6 123.9 95.8
20.3 14.9 15.1 14.1 18.6 14.5 23.5 15.8 21.2 14.6 21.8
Substrate Glycerol Methyl benzoate
73.3 125.6
33.8 20.6
99.5
25.5
1:1 molar mixture of substrates
Fig. 1. Solvent screening results on the basis of the Scatchard–Hildebrand activity coefficient: (a) pure methyl benzoate, (b) pure glycerol, and (c) glycerol + methyl benzoate (1:1 molar ratio) in the solvent mixture of acetone + 2-propanol.
15 min. Then, the lauric acid consumption was determined. One unit of activity (U) was defined as the amount of enzyme necessary to consume 1 mol of lauric acid per minute [5]. All enzymatic activity determinations were replicated at least three times. 2.3. Preliminary experiments Due to the very limited solubility exhibited by the methyl benzoate/glycerol system, at first simulation tests were accomplished to determine the most potential
G. Ceni et al. / Enzyme and Microbial Technology 46 (2010) 107–112 organic solvents in terms of substrates solubility. For this purpose, the following solvents were employed: 2-propanol, 2-methylbutanol, tert-butanol, acetone, nhexane, n-heptane, isooctane, di-isopropylether, petroleum ether, tetrahydrofuran and n-decane. The idea was to use a thermodynamic tool to guide the experimental solubility tests and help the interpretation of experimental results, thus saving time and avoiding expensive experimentation. Calculations were then performed through the activity coefficient of the Scatchard–Hildebrand regular solution theory [11] of solute (glycerol and methyl benzoate) or solute mixture at infinite dilution in pure solvent and in solvent mixtures. The parameters needed for this step were taken from Barton [12] and are presented in Table 1. Using the previous selected solvents, experimental solubility evaluation was performed by visual observation at 50 ◦ C and 150 rpm, in a mechanically stirred (IKA-RW 20 digital stirrer) jacketed glass flask (60 mL) equipped with a PT-100 probe (0.1 ◦ C accuracy) for temperature monitoring, varying the kind and amount of solvent and AOT addition (1, 5 and 10 wt%, by weight of solute mixture) in 5 mM of the pure and 1:1 molar ratio of substrates (methyl benzoate and glycerol). A second preliminary test was carried out with regard to Novozym 435 behavior in the presence of the selected pure organic solvents. This was performed at 50 ◦ C and 150 rpm, using a fixed amount of 5 mL of solvent and 5 wt% of enzyme. The enzyme was activated in oven at 40 ◦ C and 60 min. Samples were taken at regular time intervals (2, 4, 6, 8, 12, 24, 36 and 48 h) in destructive experiments, dried at 40 ◦ C for 2 h and kept by 24 h in the desiccator for later evaluation of the enzymatic activity. Then, the last preliminary test was to define the reaction time, also at 50 ◦ C and 150 rpm, using 5 mL of the selected solvent, 5 wt% of enzyme and 5 mM of substrates (1:1 molar ratio). At the same time intervals cited above, samples from destructive experiments were taken for products quantification. 2.4. Optimization of 1-glyceryl benzoate production A 22 full central composite rotatable design (CCRD) [13,14] with two axial points for each independent variable was employed in this step so as to maximize the process conversion. The variables studied were temperature (28.8–71.2 ◦ C) and enzyme concentration (1–10 wt% based on the substrates amount), keeping the reaction time constant at the value defined in the last preliminary step described before. Eleven experiments were carried out with triplicate runs performed at the central point. The mixture agitation, substrates amount and molar ratio, solvent amount and reaction time were kept constant at 150 rpm, 5 mM and 1:1, 5 mL, and 36 h, respectively. The software Statistica® 6.0 (Statsoft Inc.) was used to assist the design and the statistical analysis of experimental information, adopting in all cases studied a confidence level of 95% (p < 0.05). After analyzing the results of the experimental design, reaction kinetic experiments were performed with substrates molar ratio of 1:1, 1:2 and 1:3, enzyme concentration of 5, 10 and 15 wt% (based on the total amount of substrates), solvent amount of 3, 5 and 10 mL and temperature ranging from 40 to 70 ◦ C. It may be important to emphasize that in all cases, destructive experiments, without sampling, were carried out. After each experimental run, the enzyme was separated from the reactional medium and washed twice with 10 mL of n-hexane, following the methodology described by Castro [15]. The recuperated enzyme was kept in desiccator for 24 h and, after this period, the enzyme activity was determined. 2.5. Analysis of reaction products The reactional medium, without enzyme, was rotary-evaporated and diluted in methanol for the analysis, performed in a gas chromatography (Shimadzu model GC 17-A) equipped with a capillary column of Wcot Fused Silica (30 m × 0.32 mm) containing GP-Sil 5CB. The column was kept at 40 ◦ C by 1 min, heated to 125 ◦ C at a rate of 10 ◦ C/min (2 min) and, then, heated again to 250 ◦ C at the same rate (10 ◦ C/min). The detector and injector temperatures were kept at 300 ◦ C. The column pressure and the hydrogen flux were 8 kPa and 3.5 mL/min, respectively. The split ratio was 1:60. The structure of a standard sample of 1-glyceryl benzoate was confirmed by NMR. 1 H NMR Varian Spectra (200 MHz) in CDCl3 solution with CHCl3 as internal reference and 13 C NMR Varian Spectra (50 MHz) in CDCl3 solution with CHCl3 as internal reference were used.
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solubility, compared to methyl benzoate, in all solvents with slight better results in 2-propanol followed by tert-butanol and 2methylbutanol (Fig. 1b). The fact that acetone and 2-propanol were the best solvents for, respectively, methyl benzoate and glycerol was in fact confirmed by experimental miscibility observations at 50 ◦ C for all solvents. From these two pre-selected solvents, aiming at checking possible synergetic effect, the solubility of the substrates mixture (glycerol to methyl benzoate) at 1:1 molar ratio was estimated in the mixture formed by acetone/2-propanol. Predicted solubility results shown in Fig. 1c, confirmed by experimental observations, demonstrated that 2-propanol was the proper solvent to be used in the enzymatic reactions. Moreover, the temperature of normal boiling point of acetone is 56.1 ◦ C, making difficult its use in the enzymatic reactions in the temperature range established. One should notice the complete coherence between experiment and theory, since the Scatchard–Hildebrand relationship establishes that the closest are the solubility parameter values of solvent (or solvent mixture) and solute (or solute mixture) the smallest is the activity coefficient (gamma, approaching the unity for an ideal solution) and hence solute solubility, once from the classical thermodynamics they are inversely proportional. It is worth to mention that while 3 mL of 2-propanol was enough to reach complete solubilization of 5 mM of 1:1 methyl benzoate/glycerol, visual observations in the solubility tests with the solvents commonly used in enzymatic reactions, isooctane, n-heptane and tert-butanol demonstrated that the addition of up to 5 wt% (by weight of substrates) of AOT surfactant to the medium was required to form a macroscopic homogeneous system (thermodynamically stable reverse micelles system). Furthermore, as 2-propanol is a secondary alcohol it is hard to expect it to compete with glycerol in the enzyme-catalyzed reaction. The interaction enzyme–organic solvent was evaluated using the commercial immobilized Novozym 435 in the presence of 2propanol. The evaluation revealed no significant reduction in lipase activity after a contact time of 48 h. Based on the solubility tests presented above, 2-propanol was then chosen to carry out the enzymatic reactions. 3.1.2. Definition of reaction time Fig. 2 presents the results obtained in the transesterification of the substrates (5 mM, 1:1 molar ratio) using Novozym 435 as catalyst, at 150 rpm, 50 ◦ C and 5 mL of 2-propanol. From this figure one can observe that a reaction conversion of about 30% was
3. Results and discussion 3.1. Preliminary experiments 3.1.1. Screening of organic solvent Calculations were first performed for binary systems consisting of pure substrates and solvents. Results are shown in Fig. 1, where one can note that the Scatchard–Hildebrand relationship indicated that acetone would be the best solvent for methyl benzoate followed by (a large difference) 2-methylbutanol and tert-butanol, with relatively negligible results for other solvents (Fig. 1a). For glycerol, on the other hand, theory predicted poorer
Fig. 2. Kinetics of 1-glyceryl benzoate production using 2-propanol as solvents and Novozym 435 as catalyst. Reaction conditions: 50 ◦ C, 150 rpm, 5 mL of solvent, 5 wt% of enzyme and 5 mM of substrates (1:1 molar ratio).
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Table 2 Matrix of the 22 full CCRD experimental design for 1-glyceryl benzoate production in 2-propanol. Experimental conditions: 150 rpm, substrates amount of 5 mM (1:1 molar ratio), solvent amount of 5 mL and reaction time kept constant at 36 h. Experiment
1 2 3 4 5 6 7 8 9 10 11
Experimental condition
1-Glyceryl benzoate production
T (◦ C)
E (wt%)
Experimental (%)
Predicted (%)
35 (−1) 65 (+1) 35 (−1) 65 (+1) 28.8 (−1.41) 71.2 (+1.41) 50 (0) 50 (0) 50 (0) 50 (0) 50 (0)
2.3 (−1) 2.3 (−1) 8.7 (+1) 8.7 (+1) 5.5 (0) 5.5 (0) 1 (−1.41) 10 (+1.41) 5.5 (0) 5.5 (0) 5.5 (0)
10.1 10.5 19.2 14.5 11.2 7.0 9.5 29.6 24.4 25.4 24.9
8.7 8.7 21.7 16.5 10.6 6.9 11.8 26.5 24.9 24.9 24.9
observed at 36 h. The mass spectrum was compared to the authentic standard, confirming that the obtained product was 1-glyceryl benzoate. Based on these preliminary reaction results, the next step was performed using 2-propanol as organic solvent and a reaction time of 36 h. 3.2. Optimization of 1-glyceryl benzoate production
the experimental conversion curves versus reaction time, as presented in Fig. 4(a). From this figure one can see that an enhancement in molar ratio led to lower conversions and the use of the smallest ratio (1:1) afforded the highest conversion (approximately 30% after 80 h of reaction). It is well known that the substrates molar ratio is usually one of the most important parameters in enzymatic synthesis reactions. Since the reaction is reversible, an increase in
Results obtained from the execution of the 22 full CCRD for 1glyceryl benzoate production are presented in Table 2, where one can observe that the highest conversion (29.6%) was obtained at the experimental condition of 10 wt% of enzyme and 50 ◦ C. The statistical analysis of the data presented in Table 2 validated an empirical coded model in terms of the significant parameters, as presented by the following expression: Reaction conversion(%) = 24.9 − 1.3T − 8.1T 2 + 5.2E −2.9E 2 − 1.3TE
(1)
The empirical model permitted to build the response surface and contour curve presented in Fig. 3, showing the influence of enzyme concentration and temperature on 1-glyceryl benzoate production. The experimental condition that maximized the product conversion was obtained in the experiment 8, corresponding to 10 wt% of enzyme and 50 ◦ C, followed by the central point (5.5 wt% of enzyme and 50 ◦ C). It may be important to emphasize the good conversion values obtained in the central point with the use of relatively low enzyme concentration. 3.3. Kinetics of 1-glyceryl benzoate production Taking into account the results obtained in the experimental design, reaction kinetic experiments were performed adopting substrates molar ratios of 1:1, 1:2 and 1:3, enzyme concentration of 5, 10 and 15 wt% (based on the total amount of substrates), temperature of 50, 60 and 70 ◦ C, and solvent volume of 3, 5 and 10 mL. It is important to mention that the kinetic results presented in this work are in fact mean values of triplicate runs, which afforded an overall absolute deviation of reaction conversions of around 5%. Also, data scattering observed may be explained in terms of experimental errors associated, and the fact that destructive experiments were carried out without sampling, which may be viewed as an important internal consistence test of the results. 3.3.1. Effect of substrates molar ratio In order to evaluate the effect of glycerol to methyl benzoate molar ratio on 1-glyceryl benzoate conversion, temperature was kept fixed at 50 ◦ C, enzyme concentration at 5 wt%, 5 mL of 2propanol, 5 mM of substrates and 150 rpm, making possible to build
Fig. 3. Response surface (a) and contour curve (b) for 1-glyceryl benzoate production in terms of temperature and enzyme concentration.
G. Ceni et al. / Enzyme and Microbial Technology 46 (2010) 107–112
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Fig. 4. Kinetics of 1-glyceryl benzoate production in 2-propanol varying: (a) substrates molar ratio, (b) enzyme concentration, (c) temperature and (d) solvent amount. Reaction experiment conditions for cases (a–c): substrates amount of 5 mM, 5 mL of solvent and 150 rpm.
the concentration of one reactant can shift the reaction equilibrium to products, resulting in higher conversions. On the other hand, high methyl benzoate concentrations may reduce the reaction rate due to the inhibition effect. Based on the results obtained here, it seems that the last hypothesis had an important effect on the reaction rate. 3.3.2. Effect of enzyme concentration The effect of enzyme concentration on 1-glyceryl benzoate conversion was evaluated at 50 ◦ C keeping constant the substrates molar ratio of 1:1, 5 mL of solvent and 150 rpm, varying the enzyme concentration at 5, 10 and 15 wt% (based on the substrates amount). Fig. 4(b) shows the experimental data obtained in this step. When using 5, 10 and 15 wt% of enzyme, it can be observed that similar initial reaction rates were obtained, leading to similar conversions along all the reaction time. One may infer that the use of 5, 10 and 15 wt% of Novozym 435 did not present significant difference on 1-glyceryl benzoate production. A possible explanation for this fact might be related to the fact that an excess of enzyme in the reactional medium may not always contribute to the conversion increase, while might increase the reaction rate, since high enzyme loadings may lead to the formation of aggregates, thus not making the enzyme active site available to the substrates [16,17]. The enzyme molecules on external surface of such particles are exposed to high substrate concentrations but the mass transport could drastically limit the substrate concentration inside the particles. Lower
activities of the biocatalyst reduce the efficiency of the enzyme, not enhancing the reaction conversion [16]. 3.3.3. Effect of temperature The effect of temperature on 1-glyceryl benzoate conversion was evaluated at 50, 60 and 70 ◦ C keeping constant the substrates molar ratio at 1:1, enzyme concentration of 5 wt%, 5 mL of solvent and 150 rpm. Fig. 4(c) shows the experimental data obtained in this step. It is well known that temperature presents two important roles in this kind of reactional system. Firstly, an increase in temperature can reduce mixture viscosity, enhance mutual solubility and improve diffusion process of substrates, thus reducing mass transfer limitations and favoring interactions between enzyme particles and substrates. Further, enzymes generally have an optimal working temperature value, and in the case of Novozym 435, it is situated in the range of 40–70 ◦ C [18]. From this figure one can verify that good reaction conversions were achieved at relatively low temperatures (50 ◦ C). Finally, it may be relevant to mention that measurements of enzyme activity before (fresh) and after (used) reaction experiments (data not shown) revealed no important change in residual lipase activity at temperatures tested in this work. 3.3.4. Effect of solvent amount The effect of solvent amount on 1-glyceryl benzoate conversion was evaluated at 50 ◦ C keeping constant the substrates molar ratio
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at 1:1 (5 mM), 5 wt% of Novozym 435, 150 rpm, varying the solvent volume of 3, 5 and 10 mL. Fig. 4(d) presents the experimental data obtained in this step. It can be observed from this figure that similar initial rates were obtained when using all tested solvent volumes. Conversions of around 30% were obtained after 50 h of reaction using 5 mL of 2-propanol. After a certain period of time it seems that an excess of solvent conducted to lower conversions, probably due to losses in enzymatic activity and a lower quantity of solvent also led to lower conversions, presumably by mass transfer limitations. 4. Conclusions The use of a thermodynamic solution theory to estimate substrates solubility proved to be a simple and valuable tool for solvent screening, as visual observations of the substrates mixture confirmed 2-propanol as the best organic solvent. The use of 2-propanol conducted to 1-glyceryl benzoate conversion of approximately 30% for 36 h of reaction. It was shown in this work that it is possible to develop an enzyme-catalyzed process for 1-glyceryl benzoate production, an important intermediate for carvedilol and propranolol synthesis. Acknowledgements The authors thank FINEP, FAPERJ, CAPES and CNPq for the financial support. References [1] Kato Y, Fujiwara I, Asano Y. A novel method for preparation of optically active ␣-monobenzoyl glycerol via lipase catalyzed asymmetric transesterification of glycerol. Bioorg Med Chem Let 1999;9:3207–10.
[2] Carvalho PO, Contesini FJ, Ikegaki M. Enzymatic resolution of (R,S)-ibuprofen and (R,S)-ketoprofen by microbial lipases from native and commercial sources. Braz J Microbiol 1995;37:329–37. [3] Sih CJ, Wu SH. Resolution of enantiomers via biocatalysis. Topics in stereochemistry, asymetric synthesis. London; 1989. [4] Vedejs E, Jure MA. Efficiency in non-enzymatic kinetic resolution. Chem Int 2005;44:3974–8. [5] Oliveira D, Freire DMG, Oliveira JV, Dariva C, Feihrmann AC, Cunha AG, et al. Influence of compressed fluids treatment on the activity of Yarrowia lipolytica lipase. J Mol Catal B: Enzym 2006;39:117–23. [6] Gotor V. Non-conventional hydrolase chemistry; amide and carbamate bond formation catalyzed by lipases. Bioorg Med Chem 1999;7:2189–97. [7] Theil F. Lipase supported synthesis of biologically active compounds. Chem Rev 1995;95:2203–27. [8] Saxena RK, Davidson WS, Sheoran A, Giri B. Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus. Process Biochem 2003;39:239–47. [9] Brockerhoff HE, Jensen RG. Lipases. In: Lipolytic enzymes. 1st ed. New York: Academic Press; 1974. [10] Howe R, Rao BS. Adrenergic blocking agents. III. The optical isomers of pronethalol, propranolol and several related compounds. J Med Chem 1968;11:1118–21. [11] Prausnitz JM, Lichtenthaler RI, Gomes de Azevedo RJ. Molecular thermodynamics of fluid phase equilibria. 3rd ed. New York: Prentice Hall; 1999. [12] Barton AMF. Solubility parameters. Chem Rev 1997;75:731–53. [13] Rodrigues MI, Iemma AM. Planejamento de Experimentos e Otimizac¸ão de Processos. 1st ed. Campinas: Editora Casa do Pão; 2006. [14] Montgomery DC. Design and analysis of experiments. 1st ed. New York: John Wiley and Sons; 1991. [15] Castro HF. Fine chemicals by biotransformation using lipase. Quím Nova 1995;18:544–54. [16] Watanabe T, Shimizu M, Sugiura M, Sato M, Kohori J, Yamada N, et al. Optimization of reaction conditions for the production of DAG using immobilized 1,3-regiospecific lipase Lipozyme RM IM. J Am Oil Chem Soc 2003;80:1201–5. [17] Yang T, Rebsdorf M, Engelrud U, Xu X. Enzymatic production of monoacylglycerols containing polyunsaturated fatty acids through an efficient glycerolysis system. J Agric Food Chem 2005;53:1475–8. [18] Kristensen JB, Xu XB, Mu HB. Process optimization using response surface design and pilot plant production of dietary diacylglycerols by lipase-catalyzed glycerolysis. J Agric Food Chem 2005;53:7059–66.
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Optimization of 1-glyceryl benzoate production by enzymatic transesterification in organic solvents Giovana Ceni a,b , Lindomar A. Lerin a , Juliana Faccin de Conto b , Cristiane Vendrusculo Brancher b , Patrícia Costa da Silva b , Geciane Toniazzo b , Helen Treichel b,∗ , Débora de Oliveira b , J. Vladimir Oliveira b , Enrique Guillermo Oestreicher a , Octavio Augusto Ceva Antunes a a
Universidade Federal do Rio de Janeiro, Instituto de Química, Cidade Universitária, 21941-909, Rio de Janeiro, RJ, Brazil Universidade Regional Integrada do Alto Uruguai e das Missões (URI), Campus de Erechim Depto de Engenharia de Alimentos, Av. 7 de Setembro, 1621, 99700-000, Erechim, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 19 May 2009 Received in revised form 8 July 2009 Accepted 22 September 2009 Keywords: Kinetics Transesterification 1-Glyceryl benzoate
a b s t r a c t This work is focused on the evaluation of reaction parameters involved in the enzymatic production of 1-glyceryl benzoate, an intermediate substance to carvedilol and propranolol synthesis, catalyzed by Candida antarctica lipase in different organic solvents. To our knowledge, no related study on this subject is available in the open literature. The main goals of the present investigation were to elucidate the relationship between relevant reaction variables and also to determine the optimum conditions for 1-glyceryl benzoate production. Results showed that the strategy adopted for the experimental design proved to be useful in maximizing the reaction conversion in 2-propanol as solvent with Novozym 435 as catalyst. The optimum conditions were found to be methyl benzoate to glycerol molar ratio of 1:1, stirring rate of 150 rpm, 50 ◦ C, enzyme concentration of 10 wt% at 36 h of reaction, with a resulting conversion to 1-glyceryl benzoate of about 29%. Reaction kinetics of 1-glyceryl benzoate production demonstrated that very satisfactory conversions (∼40%) were achieved after 70 h of reaction. © 2009 Elsevier Inc. All rights reserved.
1. Introduction The use and preparation of chiral pharmaceutical principles as single enantiomers is one of the most relevant goals in pharmaceutical science. Optically active ␣-monoacyl glycerol is a useful starting material for the preparation of chiral drugs, such as blockers [1]. For chiral drugs, opposite enantioforms act with different biological properties and the distomer could give undesirable effects [2]. The resolution of racemic compounds seems to be a valuable method for obtaining chiral compounds at high optical purity [3,4] through both enzymatic [3] and non-enzymatic [4] catalysts with a wide variety of substrates. The great disadvantage of standard kinetic resolution procedure is that a maximum yield of 50% of the desired product is obtained from the starting racemic material. In this sense, enzymes have been extensively investigated for asymmetric syntheses due to the growing demand for enantiopure intermediates and drugs [3]. It is often necessary to conduct these enzymatic reactions in organic solvents towards improving substrate solubility and shifting the reaction equilibrium to products formation.
∗ Corresponding author. E-mail address: [email protected] (H. Treichel). 0141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2009.09.011
It is well known that lipases (E.C. 3.1.1.3) are very suitable enzymes for organic synthesis because they accept a wide range of non-natural substrates, are stable and active in organic solvents, do not require cofactors, and are readily available from several microorganisms. In organic solvents, lipases can be more enantioselective and the solubility of hydrophobic substrates can be enhanced [5]. Possible approaches to the enzymatic resolution of chiral products include the enantioselective hydrolysis, transesterification or the direct esterification in non-aqueous medium using microbial lipases. In this sense, lipases are today the natural choice of biocatalysts as they show unique chemo-, regio- and enatioselectivities, which enable the production of novel drugs, agrochemicals and fine chemicals [6,7]. Selection of a lipase for a given application should be based on its substrate specificity such as fatty acid, alcohol, position (regio-) and stereospecificity, as well as temperature and pH stability. Related to the huge amount and variety of applications of lipases, expected to rise in the near future, the search for lipases with characteristic substrate specificity and stability at high temperatures and in organic solvents is of great interest [8]. Because of the low solubility of many esters in some alcohols, the substrates are typically dissolved in an organic solvent, such as n-heptane. This is especially true when dealing with nonpolar organic substances and several other polar compounds such as glycerol.
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Lipases were envisaged as biocatalysts for the transesterification of methyl benzoate with glycerol to yield 1-glyceryl benzoate as main product. This compound was the obvious choice to replace epichloridrin as a green intermediate for beta-blockers production typified by carvedilol and propranolol [9]. The antagonists of -adrenergic receptors (-blockers), as propranolol [10] and carvedilol, are important active pharmaceutical ingredients (APIs) used in the treatment of diseases as hypertension, cardiopathies, some kinds of hyh and glaucoma. This class of substances has received considerable attention recently from the pharmacological and chemical point of view. In spite of the importance of producing this intermediate (1glyceryl benzoate) no experimental data on this subject was found in the current scientific literature but one paper that makes use of vinyl benzoate a much more expensive substrate [1]. It may be opportune to call attention at this point to the fact that the establishment of world-wide biodiesel government programs, with the expected huge glycerol surplus, has prompted many attempts to produce high-value added products through glycerol transformation by enzyme-catalyzed reactions. Based on these aspects, the main objective of this work was to investigate the enzymatic production of 1-glyceryl benzoate in organic solvents. Preliminary, the solubility of substrates in different organic solvents and the stability of the commercial immobilized lipase (Novozym 435) in such solvents were evaluated. From the results obtained in this step, the optimization of the process conversion and a kinetic study of enzymatic transesterification of glycerol and methyl benzoate were carried out. 2. Materials and methods 2.1. Material Glycerol and methyl benzoate (Vetec, both 99.5% purity) were used as substrates for the transesterification reactions. The commercial immobilized lipase used in this work was Candida antarctica (Novozym 435) immobilized on a macroporous anionic resin, kindly supplied by Novozymes Brazil (Araucária, PR, Brazil). The organic solvents used in the experimental tests were acetone, 2-propanol, 2-methylbutanol, tert-butanol, n-hexane, n-heptane, isooctane, di-isopropylether, petroleum ether, tetrahydrofuran and n-decane (Quimex, 99.5% purity). For some experimental conditions, sodium (bis-2-ethyl-hexyl) sulfosuccinate (aerosol-OT or AOT) was used as surfactant to allow the formation of reverse micelles. 2.2. Lipase esterification activity The enzyme activity was determined as the initial rates in esterification reactions between lauric acid and n-propanol at a molar ratio of 3:1, temperature of 60 ◦ C and enzyme concentration of 5 wt% in relation to the substrates. At the beginning of the reaction, samples containing the mixture of lauric acid and n-propanol were collected and the lauric acid content was determined by titration with NaOH 0.04N. After the addition of the enzyme to the substrates, the mixture was kept at 60 ◦ C for
Table 1 Parameters of solvents and substrates used in the solubility study [11]. Solvent
Molar volume (cm3 /g mol)
Solubility parameter (MPa)1/2
Acetone n-Hexane n-Heptane Isooctane Tetrahydrofuran Di-isopropylether 2-Propanol n-Decane 2-Methylbutanol Petroleum ether Tert-butanol
74.0 131.6 147.4 166.1 81.7 142.1 76.8 196.0 109.6 123.9 95.8
20.3 14.9 15.1 14.1 18.6 14.5 23.5 15.8 21.2 14.6 21.8
Substrate Glycerol Methyl benzoate
73.3 125.6
33.8 20.6
99.5
25.5
1:1 molar mixture of substrates
Fig. 1. Solvent screening results on the basis of the Scatchard–Hildebrand activity coefficient: (a) pure methyl benzoate, (b) pure glycerol, and (c) glycerol + methyl benzoate (1:1 molar ratio) in the solvent mixture of acetone + 2-propanol.
15 min. Then, the lauric acid consumption was determined. One unit of activity (U) was defined as the amount of enzyme necessary to consume 1 mol of lauric acid per minute [5]. All enzymatic activity determinations were replicated at least three times. 2.3. Preliminary experiments Due to the very limited solubility exhibited by the methyl benzoate/glycerol system, at first simulation tests were accomplished to determine the most potential
G. Ceni et al. / Enzyme and Microbial Technology 46 (2010) 107–112 organic solvents in terms of substrates solubility. For this purpose, the following solvents were employed: 2-propanol, 2-methylbutanol, tert-butanol, acetone, nhexane, n-heptane, isooctane, di-isopropylether, petroleum ether, tetrahydrofuran and n-decane. The idea was to use a thermodynamic tool to guide the experimental solubility tests and help the interpretation of experimental results, thus saving time and avoiding expensive experimentation. Calculations were then performed through the activity coefficient of the Scatchard–Hildebrand regular solution theory [11] of solute (glycerol and methyl benzoate) or solute mixture at infinite dilution in pure solvent and in solvent mixtures. The parameters needed for this step were taken from Barton [12] and are presented in Table 1. Using the previous selected solvents, experimental solubility evaluation was performed by visual observation at 50 ◦ C and 150 rpm, in a mechanically stirred (IKA-RW 20 digital stirrer) jacketed glass flask (60 mL) equipped with a PT-100 probe (0.1 ◦ C accuracy) for temperature monitoring, varying the kind and amount of solvent and AOT addition (1, 5 and 10 wt%, by weight of solute mixture) in 5 mM of the pure and 1:1 molar ratio of substrates (methyl benzoate and glycerol). A second preliminary test was carried out with regard to Novozym 435 behavior in the presence of the selected pure organic solvents. This was performed at 50 ◦ C and 150 rpm, using a fixed amount of 5 mL of solvent and 5 wt% of enzyme. The enzyme was activated in oven at 40 ◦ C and 60 min. Samples were taken at regular time intervals (2, 4, 6, 8, 12, 24, 36 and 48 h) in destructive experiments, dried at 40 ◦ C for 2 h and kept by 24 h in the desiccator for later evaluation of the enzymatic activity. Then, the last preliminary test was to define the reaction time, also at 50 ◦ C and 150 rpm, using 5 mL of the selected solvent, 5 wt% of enzyme and 5 mM of substrates (1:1 molar ratio). At the same time intervals cited above, samples from destructive experiments were taken for products quantification. 2.4. Optimization of 1-glyceryl benzoate production A 22 full central composite rotatable design (CCRD) [13,14] with two axial points for each independent variable was employed in this step so as to maximize the process conversion. The variables studied were temperature (28.8–71.2 ◦ C) and enzyme concentration (1–10 wt% based on the substrates amount), keeping the reaction time constant at the value defined in the last preliminary step described before. Eleven experiments were carried out with triplicate runs performed at the central point. The mixture agitation, substrates amount and molar ratio, solvent amount and reaction time were kept constant at 150 rpm, 5 mM and 1:1, 5 mL, and 36 h, respectively. The software Statistica® 6.0 (Statsoft Inc.) was used to assist the design and the statistical analysis of experimental information, adopting in all cases studied a confidence level of 95% (p < 0.05). After analyzing the results of the experimental design, reaction kinetic experiments were performed with substrates molar ratio of 1:1, 1:2 and 1:3, enzyme concentration of 5, 10 and 15 wt% (based on the total amount of substrates), solvent amount of 3, 5 and 10 mL and temperature ranging from 40 to 70 ◦ C. It may be important to emphasize that in all cases, destructive experiments, without sampling, were carried out. After each experimental run, the enzyme was separated from the reactional medium and washed twice with 10 mL of n-hexane, following the methodology described by Castro [15]. The recuperated enzyme was kept in desiccator for 24 h and, after this period, the enzyme activity was determined. 2.5. Analysis of reaction products The reactional medium, without enzyme, was rotary-evaporated and diluted in methanol for the analysis, performed in a gas chromatography (Shimadzu model GC 17-A) equipped with a capillary column of Wcot Fused Silica (30 m × 0.32 mm) containing GP-Sil 5CB. The column was kept at 40 ◦ C by 1 min, heated to 125 ◦ C at a rate of 10 ◦ C/min (2 min) and, then, heated again to 250 ◦ C at the same rate (10 ◦ C/min). The detector and injector temperatures were kept at 300 ◦ C. The column pressure and the hydrogen flux were 8 kPa and 3.5 mL/min, respectively. The split ratio was 1:60. The structure of a standard sample of 1-glyceryl benzoate was confirmed by NMR. 1 H NMR Varian Spectra (200 MHz) in CDCl3 solution with CHCl3 as internal reference and 13 C NMR Varian Spectra (50 MHz) in CDCl3 solution with CHCl3 as internal reference were used.
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solubility, compared to methyl benzoate, in all solvents with slight better results in 2-propanol followed by tert-butanol and 2methylbutanol (Fig. 1b). The fact that acetone and 2-propanol were the best solvents for, respectively, methyl benzoate and glycerol was in fact confirmed by experimental miscibility observations at 50 ◦ C for all solvents. From these two pre-selected solvents, aiming at checking possible synergetic effect, the solubility of the substrates mixture (glycerol to methyl benzoate) at 1:1 molar ratio was estimated in the mixture formed by acetone/2-propanol. Predicted solubility results shown in Fig. 1c, confirmed by experimental observations, demonstrated that 2-propanol was the proper solvent to be used in the enzymatic reactions. Moreover, the temperature of normal boiling point of acetone is 56.1 ◦ C, making difficult its use in the enzymatic reactions in the temperature range established. One should notice the complete coherence between experiment and theory, since the Scatchard–Hildebrand relationship establishes that the closest are the solubility parameter values of solvent (or solvent mixture) and solute (or solute mixture) the smallest is the activity coefficient (gamma, approaching the unity for an ideal solution) and hence solute solubility, once from the classical thermodynamics they are inversely proportional. It is worth to mention that while 3 mL of 2-propanol was enough to reach complete solubilization of 5 mM of 1:1 methyl benzoate/glycerol, visual observations in the solubility tests with the solvents commonly used in enzymatic reactions, isooctane, n-heptane and tert-butanol demonstrated that the addition of up to 5 wt% (by weight of substrates) of AOT surfactant to the medium was required to form a macroscopic homogeneous system (thermodynamically stable reverse micelles system). Furthermore, as 2-propanol is a secondary alcohol it is hard to expect it to compete with glycerol in the enzyme-catalyzed reaction. The interaction enzyme–organic solvent was evaluated using the commercial immobilized Novozym 435 in the presence of 2propanol. The evaluation revealed no significant reduction in lipase activity after a contact time of 48 h. Based on the solubility tests presented above, 2-propanol was then chosen to carry out the enzymatic reactions. 3.1.2. Definition of reaction time Fig. 2 presents the results obtained in the transesterification of the substrates (5 mM, 1:1 molar ratio) using Novozym 435 as catalyst, at 150 rpm, 50 ◦ C and 5 mL of 2-propanol. From this figure one can observe that a reaction conversion of about 30% was
3. Results and discussion 3.1. Preliminary experiments 3.1.1. Screening of organic solvent Calculations were first performed for binary systems consisting of pure substrates and solvents. Results are shown in Fig. 1, where one can note that the Scatchard–Hildebrand relationship indicated that acetone would be the best solvent for methyl benzoate followed by (a large difference) 2-methylbutanol and tert-butanol, with relatively negligible results for other solvents (Fig. 1a). For glycerol, on the other hand, theory predicted poorer
Fig. 2. Kinetics of 1-glyceryl benzoate production using 2-propanol as solvents and Novozym 435 as catalyst. Reaction conditions: 50 ◦ C, 150 rpm, 5 mL of solvent, 5 wt% of enzyme and 5 mM of substrates (1:1 molar ratio).
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Table 2 Matrix of the 22 full CCRD experimental design for 1-glyceryl benzoate production in 2-propanol. Experimental conditions: 150 rpm, substrates amount of 5 mM (1:1 molar ratio), solvent amount of 5 mL and reaction time kept constant at 36 h. Experiment
1 2 3 4 5 6 7 8 9 10 11
Experimental condition
1-Glyceryl benzoate production
T (◦ C)
E (wt%)
Experimental (%)
Predicted (%)
35 (−1) 65 (+1) 35 (−1) 65 (+1) 28.8 (−1.41) 71.2 (+1.41) 50 (0) 50 (0) 50 (0) 50 (0) 50 (0)
2.3 (−1) 2.3 (−1) 8.7 (+1) 8.7 (+1) 5.5 (0) 5.5 (0) 1 (−1.41) 10 (+1.41) 5.5 (0) 5.5 (0) 5.5 (0)
10.1 10.5 19.2 14.5 11.2 7.0 9.5 29.6 24.4 25.4 24.9
8.7 8.7 21.7 16.5 10.6 6.9 11.8 26.5 24.9 24.9 24.9
observed at 36 h. The mass spectrum was compared to the authentic standard, confirming that the obtained product was 1-glyceryl benzoate. Based on these preliminary reaction results, the next step was performed using 2-propanol as organic solvent and a reaction time of 36 h. 3.2. Optimization of 1-glyceryl benzoate production
the experimental conversion curves versus reaction time, as presented in Fig. 4(a). From this figure one can see that an enhancement in molar ratio led to lower conversions and the use of the smallest ratio (1:1) afforded the highest conversion (approximately 30% after 80 h of reaction). It is well known that the substrates molar ratio is usually one of the most important parameters in enzymatic synthesis reactions. Since the reaction is reversible, an increase in
Results obtained from the execution of the 22 full CCRD for 1glyceryl benzoate production are presented in Table 2, where one can observe that the highest conversion (29.6%) was obtained at the experimental condition of 10 wt% of enzyme and 50 ◦ C. The statistical analysis of the data presented in Table 2 validated an empirical coded model in terms of the significant parameters, as presented by the following expression: Reaction conversion(%) = 24.9 − 1.3T − 8.1T 2 + 5.2E −2.9E 2 − 1.3TE
(1)
The empirical model permitted to build the response surface and contour curve presented in Fig. 3, showing the influence of enzyme concentration and temperature on 1-glyceryl benzoate production. The experimental condition that maximized the product conversion was obtained in the experiment 8, corresponding to 10 wt% of enzyme and 50 ◦ C, followed by the central point (5.5 wt% of enzyme and 50 ◦ C). It may be important to emphasize the good conversion values obtained in the central point with the use of relatively low enzyme concentration. 3.3. Kinetics of 1-glyceryl benzoate production Taking into account the results obtained in the experimental design, reaction kinetic experiments were performed adopting substrates molar ratios of 1:1, 1:2 and 1:3, enzyme concentration of 5, 10 and 15 wt% (based on the total amount of substrates), temperature of 50, 60 and 70 ◦ C, and solvent volume of 3, 5 and 10 mL. It is important to mention that the kinetic results presented in this work are in fact mean values of triplicate runs, which afforded an overall absolute deviation of reaction conversions of around 5%. Also, data scattering observed may be explained in terms of experimental errors associated, and the fact that destructive experiments were carried out without sampling, which may be viewed as an important internal consistence test of the results. 3.3.1. Effect of substrates molar ratio In order to evaluate the effect of glycerol to methyl benzoate molar ratio on 1-glyceryl benzoate conversion, temperature was kept fixed at 50 ◦ C, enzyme concentration at 5 wt%, 5 mL of 2propanol, 5 mM of substrates and 150 rpm, making possible to build
Fig. 3. Response surface (a) and contour curve (b) for 1-glyceryl benzoate production in terms of temperature and enzyme concentration.
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Fig. 4. Kinetics of 1-glyceryl benzoate production in 2-propanol varying: (a) substrates molar ratio, (b) enzyme concentration, (c) temperature and (d) solvent amount. Reaction experiment conditions for cases (a–c): substrates amount of 5 mM, 5 mL of solvent and 150 rpm.
the concentration of one reactant can shift the reaction equilibrium to products, resulting in higher conversions. On the other hand, high methyl benzoate concentrations may reduce the reaction rate due to the inhibition effect. Based on the results obtained here, it seems that the last hypothesis had an important effect on the reaction rate. 3.3.2. Effect of enzyme concentration The effect of enzyme concentration on 1-glyceryl benzoate conversion was evaluated at 50 ◦ C keeping constant the substrates molar ratio of 1:1, 5 mL of solvent and 150 rpm, varying the enzyme concentration at 5, 10 and 15 wt% (based on the substrates amount). Fig. 4(b) shows the experimental data obtained in this step. When using 5, 10 and 15 wt% of enzyme, it can be observed that similar initial reaction rates were obtained, leading to similar conversions along all the reaction time. One may infer that the use of 5, 10 and 15 wt% of Novozym 435 did not present significant difference on 1-glyceryl benzoate production. A possible explanation for this fact might be related to the fact that an excess of enzyme in the reactional medium may not always contribute to the conversion increase, while might increase the reaction rate, since high enzyme loadings may lead to the formation of aggregates, thus not making the enzyme active site available to the substrates [16,17]. The enzyme molecules on external surface of such particles are exposed to high substrate concentrations but the mass transport could drastically limit the substrate concentration inside the particles. Lower
activities of the biocatalyst reduce the efficiency of the enzyme, not enhancing the reaction conversion [16]. 3.3.3. Effect of temperature The effect of temperature on 1-glyceryl benzoate conversion was evaluated at 50, 60 and 70 ◦ C keeping constant the substrates molar ratio at 1:1, enzyme concentration of 5 wt%, 5 mL of solvent and 150 rpm. Fig. 4(c) shows the experimental data obtained in this step. It is well known that temperature presents two important roles in this kind of reactional system. Firstly, an increase in temperature can reduce mixture viscosity, enhance mutual solubility and improve diffusion process of substrates, thus reducing mass transfer limitations and favoring interactions between enzyme particles and substrates. Further, enzymes generally have an optimal working temperature value, and in the case of Novozym 435, it is situated in the range of 40–70 ◦ C [18]. From this figure one can verify that good reaction conversions were achieved at relatively low temperatures (50 ◦ C). Finally, it may be relevant to mention that measurements of enzyme activity before (fresh) and after (used) reaction experiments (data not shown) revealed no important change in residual lipase activity at temperatures tested in this work. 3.3.4. Effect of solvent amount The effect of solvent amount on 1-glyceryl benzoate conversion was evaluated at 50 ◦ C keeping constant the substrates molar ratio
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at 1:1 (5 mM), 5 wt% of Novozym 435, 150 rpm, varying the solvent volume of 3, 5 and 10 mL. Fig. 4(d) presents the experimental data obtained in this step. It can be observed from this figure that similar initial rates were obtained when using all tested solvent volumes. Conversions of around 30% were obtained after 50 h of reaction using 5 mL of 2-propanol. After a certain period of time it seems that an excess of solvent conducted to lower conversions, probably due to losses in enzymatic activity and a lower quantity of solvent also led to lower conversions, presumably by mass transfer limitations. 4. Conclusions The use of a thermodynamic solution theory to estimate substrates solubility proved to be a simple and valuable tool for solvent screening, as visual observations of the substrates mixture confirmed 2-propanol as the best organic solvent. The use of 2-propanol conducted to 1-glyceryl benzoate conversion of approximately 30% for 36 h of reaction. It was shown in this work that it is possible to develop an enzyme-catalyzed process for 1-glyceryl benzoate production, an important intermediate for carvedilol and propranolol synthesis. Acknowledgements The authors thank FINEP, FAPERJ, CAPES and CNPq for the financial support. References [1] Kato Y, Fujiwara I, Asano Y. A novel method for preparation of optically active ␣-monobenzoyl glycerol via lipase catalyzed asymmetric transesterification of glycerol. Bioorg Med Chem Let 1999;9:3207–10.
[2] Carvalho PO, Contesini FJ, Ikegaki M. Enzymatic resolution of (R,S)-ibuprofen and (R,S)-ketoprofen by microbial lipases from native and commercial sources. Braz J Microbiol 1995;37:329–37. [3] Sih CJ, Wu SH. Resolution of enantiomers via biocatalysis. Topics in stereochemistry, asymetric synthesis. London; 1989. [4] Vedejs E, Jure MA. Efficiency in non-enzymatic kinetic resolution. Chem Int 2005;44:3974–8. [5] Oliveira D, Freire DMG, Oliveira JV, Dariva C, Feihrmann AC, Cunha AG, et al. Influence of compressed fluids treatment on the activity of Yarrowia lipolytica lipase. J Mol Catal B: Enzym 2006;39:117–23. [6] Gotor V. Non-conventional hydrolase chemistry; amide and carbamate bond formation catalyzed by lipases. Bioorg Med Chem 1999;7:2189–97. [7] Theil F. Lipase supported synthesis of biologically active compounds. Chem Rev 1995;95:2203–27. [8] Saxena RK, Davidson WS, Sheoran A, Giri B. Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus. Process Biochem 2003;39:239–47. [9] Brockerhoff HE, Jensen RG. Lipases. In: Lipolytic enzymes. 1st ed. New York: Academic Press; 1974. [10] Howe R, Rao BS. Adrenergic blocking agents. III. The optical isomers of pronethalol, propranolol and several related compounds. J Med Chem 1968;11:1118–21. [11] Prausnitz JM, Lichtenthaler RI, Gomes de Azevedo RJ. Molecular thermodynamics of fluid phase equilibria. 3rd ed. New York: Prentice Hall; 1999. [12] Barton AMF. Solubility parameters. Chem Rev 1997;75:731–53. [13] Rodrigues MI, Iemma AM. Planejamento de Experimentos e Otimizac¸ão de Processos. 1st ed. Campinas: Editora Casa do Pão; 2006. [14] Montgomery DC. Design and analysis of experiments. 1st ed. New York: John Wiley and Sons; 1991. [15] Castro HF. Fine chemicals by biotransformation using lipase. Quím Nova 1995;18:544–54. [16] Watanabe T, Shimizu M, Sugiura M, Sato M, Kohori J, Yamada N, et al. Optimization of reaction conditions for the production of DAG using immobilized 1,3-regiospecific lipase Lipozyme RM IM. J Am Oil Chem Soc 2003;80:1201–5. [17] Yang T, Rebsdorf M, Engelrud U, Xu X. Enzymatic production of monoacylglycerols containing polyunsaturated fatty acids through an efficient glycerolysis system. J Agric Food Chem 2005;53:1475–8. [18] Kristensen JB, Xu XB, Mu HB. Process optimization using response surface design and pilot plant production of dietary diacylglycerols by lipase-catalyzed glycerolysis. J Agric Food Chem 2005;53:7059–66.