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Synthesis of esters by immobilized-lipase-catalyzed condensation reaction of sugars and fatty acids in water-miscible organic solvent
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 99, No. 2, 87–94. 2005 DOI: 10.1263/jbb.99.087
© 2005, The Society for Biotechnology, Japan
Synthesis of Esters by Immobilized-Lipase-Catalyzed Condensation Reaction of Sugars and Fatty Acids in Water-Miscible Organic Solvent SHUJI ADACHI1* AND TAKASHI KOBAYASHI2 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan1 and Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan2 Received 25 October 2004/Accepted 1 December 2004
A lipase-catalyzed condensation reaction in an organic solvent is a promising means of synthesizing esters. Reaction equilibrium constant, which is usually defined on the basis of reactant concentration, is an important parameter for estimating equilibrium yield. It is shown that the constant is markedly, affected by some factors, such as the hydration of a sugar substrate and the interaction of a reactant with a solvent. To reasonably design the reaction system or determine the reaction conditions, attention should be paid to these factors. From the viewpoint of kinetics, substrate selectivity for carboxylic acids also numerically correlates to the electrical and steric properties of these acids. Reactor systems for continuously producing esters through an immobilizedlipase-catalyzed condensation reaction are developed. [Key words: lipase, condensation, reaction equilibrium, ester, organic solvent]
under any conditions. However, only a few studies have shown the factors affecting such a constant for lipase-catalyzed condensation reaction in organic solvents. Most of investigations on lipase-catalyzed condensation reactions have been carried out using a small-scale batch reactor, although a continuous reaction is preferred for the large-scale production of esters. In this context, the factors affecting the reaction equilibrium constant will first be described, and the substrate se-
Esters are used in many foods, cosmetics, medicines and chemicals. Compared with conventional chemical syntheses, enzymatic preparation has some advantages, such as onestep synthesis without the protection and deprotection of polyols (1) and moderate reaction conditions. A lipase (EC 3.1.1.3) catalyzes a condensation reaction (reverse hydrolysis) to produce various esters, such as aliphatic alcohol esters (2–7), hydroxy fatty acid esters (8), polyesters (9, 10) and polyol esters (Table 1). Lipases are also widely used in various reactions, such as transesterification and alcoholysis, for the production of optically active compounds (11–17) and biodiesel fuel (18–20) as well as for the breakdown of fats and oil. The customization of lipases for practical use is extensively reviewed (21). In this study, the syntheses of esters by a lipase-catalyzed condensation reaction in watermiscible organic-solvent systems will be investigated. A hydrolase-catalyzed reaction in a conventional aqueous system thermodynamically favors hydrolysis. Because a lipase-catalyzed reaction is also such a case, a condensation reaction to produce an ester is carried out in a nonaqueous medium such as an organic solvent, a solvent-free system or an ionic liquid (22). Some lipases have catalytic activity even in the presence of small amounts of water (23). Reaction equilibrium constant is a crucial parameter in the prediction of the equilibrium yield of a desired product
TABLE 1. Synthesis of esters by lipase-catalyzed condensation reaction in organic solvent systems Alcohol substrate Glucose Glucose Fructose Mannose Galactose Methyl-a-D-glucoside Butyl-a-D-glucoside Octyl-a-D-glucoside Hydroxypropyl cellulose Glycerol Erythritol Xylitol Sorbitol Ribitol Ascorbic acid Kojic acid
* Corresponding author. e-mail: [email protected] phone: +81-(0)75-753-6286 fax: +81-(0)75-753-6285 87
Acid substrate Fatty acid Vinylacetic acid Fatty acid Fatty acid Fatty acid 1-Bromomyristic acid Fatty acid Cinnamic acid Fatty acid Fatty acid Fatty acid Fatty acid Fatty acid Fatty acid Fatty acid Fatty acid
References 1, 40, 41, 66, 67 45 36, 68 44, 59 60 69 70 71 72 37, 42, 73 56, 74 43 75 58 38, 64, 65, 76–82 52, 83, 84
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lectivity of a lipase for various carboxylic acids will follow. Finally, reactor systems for the continuous production of esters will be mentioned. The results obtained using only the lipase from Candida antarctica fraction B will be shown. CONDENSATION AND TRANSESTERIFICATION An ester can be synthesized through the lipase-catalyzed condensation of a carboxylic acid and an alcohol. Esters can also be produced by lipase-catalyzed transestrifications, which can be classified into alcoholysis, acidolysis and interesterification. In an organic solvent, alcoholysis uses methyl, ethyl and vinyl esters as substrates. Vinyl ester is most extensively used among esters because its by-product, vinyl alcohol, is almost irreversibly converted to acetaldehyde, thereby increasing the yield of the desired ester (24–30). Such conversion is generally higher in transesterification than in condensation (7, 31). However, the Schiff-base formation of acetaldehyde with lipase might present a problem (32). REACTION EQUILIBRIUM CONSTANT Definition of reaction equilibrium constant The condensation of an alcohol, A, and a carboxylic (or fatty) acid, F, results in the formation of a product (an ester), P, and a by-product (water), W. A+F
P+W
(1)
Reaction equilibrium constant, KC, is usually defined on the basis of the equilibrium concentrations, C, of the reactants because of its convenience in estimating the equilibrium yield of a desired product. KC = CPCW/(CACF)
(2)
However, reaction equilibrium constant, Ka, is thermodynamically defined on the basis of the activities, a, of the reactants by Ka = aPaW/(aAaF)
FIG. 1. Effects of initial water content on equilibrium yields of lauroyl glucose (closed circles), galactose (closed diamonds), mannose (closed squares) and fructose (closed triangles) and solubilities at 50°C of glucose (open circles), galactose (open diamonds), mannose (open squares) and fructose (open triangles) in acetonitrile (40).
this reaction system, the unreacted hexose remained in the solid state. To estimate the equilibrium concentration of each hexose, the solubility of the hexose in the solvent was measured at various water contents. The solubility could empirically be expressed as an exponential function of water content. The KC values for the synthesis of the lauroyl hexoses were evaluated. KC significantly depended on the type of hexose even though the bond formed by a condensation reaction is common for every hexose. As a possible reason for the dependence, the hydration of hexose was considered to affect equilibrium conversion. The hexose that is more strongly hydrated further decreased the water activity in the system to shift the equilibrium toward synthesis. The dynamic hydration number of hexose (34, 35) was selected as a measure of the extent of hydration. As shown in Fig. 2, there was a positive correlation between KC and the dynamic hy-
(3)
Ka is intrinsic under specific temperature and pressure, while KC is apparent because it is affected by environmental conditions. Although there is an attempt to estimate a with the UNIFAC (33), it is either impossible or extremely difficult to evaluate all the a values of the reactants. This is also a reason for the convenient use of KC. Ka and KC are related to each other through the activity coefficients, g, of the reactants by gA gF KC = -----------K (4) gP gW a Effects of water on reaction equilibrium Hydration of a saccharide Figure 1 shows the equilibrium conversions of lauroyl glucose, galactose, mannose and fructose in acetonitrile with various initial water contents. The equilibrium conversion was low at a high water content in every solvent, though this tendency is reasonable because water is one of the products of condensation. The term conversion indicates the molar ratio of a desired product to a limiting substrate, hexose (generally, alcohol substrate). In
FIG. 2. Correlations of the apparent equilibrium constant KC for lauroyl hexose synthesis in acetonitrile with the dynamic hydration number of hexoses (closed squares) (40), and of KC for lauroyl mannose synthesis with relative dielectric constant of various solvents (open squares) (44). 2M2P, 2-Methyl-2-propanol; 2M2B, 2-methyl2-butanol.
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FIG. 3. Equilibrium conversion for lauroyl mannose synthesis in 2-methyl-2-propanol (circles) and 2-methyl-2-butanol (triangles) at various amounts of molecular sieve 3A (39). Inset: adsorption isotherms at 50°C of water onto molecular sieve 3A in the solvents.
dration number, indicating that water activity plays an important role in the condensation reaction in organic solvents. Condensation in the presence of desiccant Water is formed by a condensation reaction. Its removal from a reaction system is effective in shifting the reaction toward ester formation. For this reason, the addition of a desiccant (i.e., a molecular sieves [1, 31, 36–38] or silica gel [5, 10]) to a reaction system has often been adopted. However, there is as yet no criterion for the amount of desiccant to be added to achieve the desired conversion. A method of predicting equilibrium conversion for monoacyl hexose synthesis by a lipase-catalyzed condensation reaction in a water-miscible solvent in the presence of a molecular sieve has been proposed. The method is based on the adsorption isotherm of water onto molecular sieves, KC, the solubility of the hexose in the solvent, and the mass balance of water. Figure 3 shows the apparent equilibrium conversions of mannose and lauric acid to lauroyl mannose by an immobilized lipase in 2-methyl-2-propanol and 2-methyl-2-butanol in the presence of various amounts of molecular sieve 3A (39). The conversion was high in the presence of large amounts of the molecular sieve. The solid curves in Fig. 3 were calculated by the proposed method and expressed well the experimental results. The inset of Fig. 3 shows the adsorption isotherms at 50°C of water onto molecular sieve 3A in the solvents. The isotherms are expressed by the Langmuir equation. The molecular sieve acts as a catalyst as well as an adsorbent. There are some cases in which undesirable reactions, such as the degradation of unstable substrates and diester formation, proceed (10, 36, 38). Solvent effects on the reaction equilibrium Both water-miscible and -immiscible organic solvents have been used as reaction media for lipase-catalyzed condensation reactions. The use of water-miscible solvents, such as acetone, acetonitrile and tertiary alcohols, has the advantage that hydrophilic substrates, such as saccharides, are solubilized to some extent without the addition of solubilizing reagents to facilitate the esterification of the substrates (1, 40– 43). However, a water-miscible solvent removes water from
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a lipase molecule, which is essential for catalytic activity; the removal might bring about the deactivation of the lipase. A condensation reaction is usually performed in a pure solvent with a low water content. Even though the solvent provides only the field of the reaction, there are some cases in which the type of solvent affects equilibrium conversion. Changing the solvent from hexane to tertiary alcohols suppresses diester (triester) formation preceding monoester formation for the synthesis of oleoyl xylitols (43). The difference in equilibrium conversion was discussed on the basis of the polarity of the solvent. Lauroyl mannose (44) and vinylacetyl glucose (45) were synthesized by a lipase-catalyzed condensation reactions in various water-miscible solvents. The KC values for lauroyl mannose formation differed by about two orders of magnitude among the solvents. The KC values were the highest in acetonitrile, intermediate in acetone, and low in 2-methyl-2-propanol and in 2-methyl2-butanol. A log P value, where P is the partition coefficient between 1-octanol and water phases, is most frequently used in characterizing a solvent (4, 46). An empirical DimrothReichardt parameter, ET(30), expressing the polarity of a solvent (47) is also used in assessing the enzyme stability in organic solvents (48). The correlations of KC with log P, ET(30) and the relative dielectric constant of the solvent were examined. KC (ln KC) correlated best with the relative dielectric constant as shown in Fig. 2 (44). However, the dielectric constant was not applicable in analyzing the formation of butyl decanoate (6). Therefore, the correlation obtained for lauroyl mannose synthesis seemed to be apparent, as will be discussed later. A pure organic solvent with inherent properties is usually used as a reaction medium. The mixing of two different solvents can adjust the values of the properties between their inherent values, and allows us to examine the solvent effect on reaction equilibrium in detail. Oleoyl xylitols were synthesized in mixed solvents and the equilibrium conversion to a diester was found to increase with decreasing ET(30) (43). KC for butyl decanoate synthesis was estimated in mix-
FIG. 4. Apparent equilibrium constant for butyl decanoate synthesis in mixtures of a tertiary alcohol and a nitrile at various ratios. The open and closed symbols represent the mixtures of 2-methyl-2-propanol and 2-methyl-2-butanol, respectively, with acetonitrile (diamonds), propionitrile (squares) and butyronitrile (triangles) (6).
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TABLE 2. Carboxylic acids used for examination of substrate selectivity Non-conjugated Conjugated
Substrate no. 1 2 3 7 8
Straight chain Propionic acid Butyric acid Vinylacetic acid Acrylic acid Crotonic acid
tures of a nitrile and a tertiary alcohol at various ratios (6). As shown in Fig. 4, KC increased with the fraction of nitrile. The alkyl chain length of the nitrile or tertiary alcohol only slightly affected KC. These results suggest that the reaction equilibrium can be, to a certain degree, controlled using a mixture of a nitrile and a tertiary alcohol as the reaction medium, and that the polar region of a solvent molecule markedly affects KC. The infrared (IR) spectra of decanoic acid and butyl decanoate were measured in mixtures of a nitrile and a tertiary alcohol. The IR absorption peak of C = O for decanoic acid shifted to a higher wave number at a higher fraction of acetonitrile in the mixture with 2-methyl-2-propanol. The change in wave number for butyl decanoate was smaller than that for decanoic acid. The KC estimated for the mixture of acetonitrile and 2-methyl-2-propanol, on a semi-logarithmic scale, could linearly correlate to wave number at the peak of the C= O double bond of decanoic acid. These results indicate that decanoic acid (fatty acid) interacts more strongly with 2-methyl-2-propanol (a tertiary alcohol) than with butyl decanoate (ester).
Substrate no. 4 5 6 9 10
Branched chain Isobutyric acid Isovaleric acid Cyclohexanecarboxylic acid Methacrylic acid Benzoic acid
also, on a semi-logarithmic scale, plotted against the projection area. The plots for the conjugated acids with straight and branched chains form a straight line, and those for the nonconjugated acids form another straight line. The two lines are almost parallel. These results roughly indicate that the presence of a conjugation or a branched chain independently lowers Vmax/Km by one order; presence of both decreases Vmax/Km by two orders. REACTORS FOR CONTINUOUS SYNTHESIS OF ESTERS A batch reactor is generally used for the lipase-catalyzed
SUBSTRATE SELECTIVITY FOR CONDENSATION REACTION There are some cases in which a condensation reaction proceeds rather slowly, e.g., the synthesis of ferulic acid esters (7). Although the electrical and steric effects of substrates on reaction rate were reported (49, 50), no attempt was made to numerically correlate the effects with substrate selectivity. The maximum reaction rate Vmax and the Michaelis constant Km were evaluated for the condensation reactions of various short-chain carboxylic acids with p-methoxyphenethyl alcohol (51). The carboxylic acids used as the substrate can be classified into four groups by the presence of branched chains and conjugations in their molecular structures (Table 2). The steric and electrical properties of the substrate molecules were estimated from the optimized structure of a carboxylic acid molecule and the electron density of a carboxylic carbon using the molecular orbital calculation software MOPAC 2000. Figure 5 shows the relationship between Vmax/Km and the electron density of a carboxyl carbon of an acid. The Vmax/Km values of the nonconjugated acids were about 10 times higher than those of the conjugated ones; those of the acids with straight chains were also about 10 times higher than those of the acids with branched chains. Figure 6 shows the relationship between the projection area of the non-carboxylic region of an acid and the Km of the acid. The Km is linearly related to the projection area, indicating that a more bulky acid exhibits a greater steric hindrance to the formation of the enzyme-substrate complex. The Vmax/Km is
FIG. 5. Relationship between log Vmax/Km of acid and electron density of carboxyl carbon of acid (51). Vmax/Km is expressed in min–1. Labels in the graph correspond to the substrate number in Table 2.
FIG. 6. Effects of projection area of non-carboxylic region of acid molecule on log Vmax/Km and Km of acid (51). Vmax/Km is expressed in min–1. Labels in the graph correspond to the substrate number in Table 2.
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FIG. 7. Continuous syntheses of oleoyl (squares), linoleoyl (reverse triangles), decanoyl (circles), lauroyl (diamonds) and myristoyl (triangles) L-ascorbates using reactor system illustrated in graph (55). 1, Feed reservoir; 2, pump; 3, preheating coil; 4, column packed with ascorbic acid powders; 5, column packed with immobilized lipase; 6, thermoregulated chamber; 7, effluent reservoir.
synthesis of esters, although a continuous reactor would be preferred for their large-scale production. Continuous reactors are typically classified into a continuous stirred tank reactor (CSTR) and a plug flow reactor (PFR) on the basis of the type of liquid mixing in the reactor. Because the solubility of an alcohol substrate, such as saccharide, in an organic solvent is low, the substrate remains undissolved in the solvent when an excess amount of the substrate is added. Therefore, CSTR seems to be a candidate system for the continuous production of an ester through a lipase-catalyzed condensation reaction in a solvent. We realized the continuous or semicontinuous syntheses of fatty acid esters of kojic acid (52), maltose (53) and ascorbic acid (54) using CSTR. The solubility of an alcohol substrate in an organic solvent generally ranges from 1 to 50 mmol/l. For a product, if we assume that its conversion is 0.7 and its molecular mass is 300, its concentration range 0.2–10 g/l. This concentration is not high, but is not extremely low either. Based on such a hypothetical calculation, a PFR system for the continuous production of an ester was proposed, as shown in the inset of Fig. 7. Powders of an alcohol substrate were packed into a column, immobilized-lipase particles were packed into another column, and the columns were then connected in series. A fatty acid solution dissolved in an organic solvent was fed to the alcohol substrate column to dissolve the substrate to a concentration close to saturation, and then pumped to the lipase column to produce an ester. Figure 7 shows the continuous synthesis of various acyl ascorbates using such a reactor system (55). Each acyl ascorbate was produced for 2 d. The system could be stably operated for at least 10 d, and the product concentrations in the effluent were in the range of 14 to 17 mmol/l. These product concentrations corresponded to the productivity of 1.6 to 1.9 kg/l-reactor/d, depending on the molecular mass of the product.
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FIG. 8. Oxidation of a-linolenic (open reverse triangles), eicosapentaenoic (open circles) and docosahexaenoic (open diamond) acids, and a-linolenoyl (closed reverse triangles), eicosapentaenoyl (closed circles) and docosahexaenoyl (closed diamonds) L-ascorbates at 65°C and almost 0% relative humidity (64, 65).
The PFR system was successfully applied to the continuous synthesis of fatty acid esters of erythritol (56, 57), various sugar alcohols (58) and hexoses (59, 60). The properties of the esters synthesized through a lipasecatalyzed condensation reaction are described in terms of their applications. As an example, the properties of saturated or unsaturated acyl ascorbate will briefly be described. The radical scavenging activity of ascorbic acid is not affected by the introduction of an acyl group to the hydroxyl group at the C-6 position of the ascorbic acid (54). Although ascorbic acid is insoluble in soybean oil, the acylation of ascorbic acid largely improves its solubility in the oil (54). Saturated acyl ascorbates were surface-active and their critical micelle concentrations were measured (61). The acyl ascorbates can be used in the microencapsulation of an unsaturated fatty acid with a polysaccharide by spray drying and can effectively suppress the oxidation of the encapsulated fatty acid (62). The ascorbates were also effective for suppressing the oxidation of the membrane of intestinal epithelial cells (63). Polyunsaturated fatty acids are prone to oxidation. Their esters with ascorbic acid are very resistant to oxidation. Figure 8 shows the oxidation processes of the unmodified a-linolenic, eicosapentaenoic and docosahexaenoic acids and the acyl moiety of their ascorbates at 65°C and almost 0% relative humidity (64, 65). The unmodified fatty acids were completely oxidized within 2 h, while all the unsaturated acyl moieties of the ascorbates remained unoxidized in that period. It was also shown that the addition of a saturated acyl ascorbate to an unsaturated fatty acid significantly suppresses the oxidation of the unsaturated fatty acid (65). REFERENCES 1. Arcos, J. A., Bernabé, M., and Otero, C.: Quantitative enzymatic production of 6-O-acylglucose esters. Biotechnol.
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the synthesis of acyl hexose through lipase-catalyzed condensation in water-miscible solvent in the presence of molecular sieve. Biotechnol. Prog., 19, 293–297 (2003). Watanabe, Y., Miyawaki, Y., Adachi, S., Nakanishi, K., and Matsuno, R.: Synthesis of lauroyl saccharides through lipase-catalyzed condensation in microaqueous water-miscible solvents. J. Mol. Catal. B: Enzym., 10, 241–247 (2000). Degn, P. and Zimmermann, W.: Optimization of carbohydrate fatty acid ester synthesis in organic media by a lipase from Candida antarctica. Biotechnol. Bioeng., 74, 483–491 (2001). Otero, C., Arcos, J. A., Berrendero, M. A., and Torres, C.: Emulsifiers from solid and liquid polyols: different strategies for obtaining optimum conversions and selectivities. J. Mol. Catal. B: Enzym., 11, 883–892 (2001). Castillo, E., Pezzotti, F., Navarro, A., and López-Munguía, A.: Lipase-catalyzed synthesis of xylitol monoesters: solvent engineering approach. J. Biotechnol., 102, 251–259 (2003). Watanabe, Y., Miyawaki, Y., Adachi, S., Nakanishi, K., and Matsuno, R.: Equilibrium constant for lipase-catalyzed condensation of mannose and lauric acid in water-miscible organic solvents. Enzyme Microb. Technol., 29, 494–498 (2001). Zhang, X., Kobayashi, T., Adachi, S., and Matsuno, R.: Lipase-catalyzed synthesis of 6-O-vinylacetyl glucose in acetonitrile. Biotechnol. Lett., 24, 1097–1100 (2002). Tewari, Y. B., Schantz, M. M., and Vanderah, D. J.: Thermodynamics of the lipase-catalyzed esterification of 1-dodecanoic acid with (-)-menthol in organic solvents. J. Chem. Eng. Data, 44, 641–647 (1999). Reichardt, C.: Empirical parameters of solvent polarity as linear free-energy relationships. Angew. Chem. Int. Ed. Engl., 18, 98–110 (1979). Miyanaga, M., Ohmori, M., Imamura, K., Sakiyama, T., and Nakanishi, K.: Stability of immobilized thermolysin in organic solvents. J. Biosci. Bioeng., 87, 463–472 (1999). Charton, M.: Contributions of steric, electrical, and polarizability effects in enantioselective hydrolyses with Rhizopus nigricans: a quantitative analysis. J. Org. Chem., 52, 2400– 2403 (1987). Bevinakatti, H. S. and Banerji, A. A.: Lipase catalysis: factors governing transesterification. Biotechnol. Lett., 6, 397– 398 (1988). Kobayashi, T., Adachi, S., and Matsuno, R.: Lipase-catalyzed condensation of p-methoxyphenethyl alcohol and carboxylic acids with different steric and electrical properties in acetonitrile. Biotechnol. Lett., 25, 3–7 (2003). Kobayashi, T., Adachi, S., Nakanishi, K., and Matsuno, R.: Semi-continuous production of lauroyl kojic acid through lipase-catalyzed condensation in acetonitrile. Biochem. Eng. J., 9, 85–89 (2001). Zhang, X., Kobayashi, T., Watanabe, Y., Fujii, T., Adachi, S., Nakanishi, K., and Matsuno, R.: Lipase-catalyzed synthesis of monolauroyl maltose through condensation of maltose and lauric acid. Food Sci. Technol. Res., 9, 110–113 (2003). Watanabe, Y., Kuwabara, K., Adachi, S., Nakanishi, K., and Matsuno, R.: Production of saturated acyl L-ascorbate by immobilized lipase using a continuous stirred tank reactor. J. Agric. Food Chem., 51, 4628–4632 (2003). Kuwabara, K., Watanabe, Y., Adachi, S., Nakanishi, K., and Matsuno, R.: Continuous production of acyl L-ascorbates using a packed-bed reactor with immobilized lipase. J. Am. Oil Chem. Soc., 80, 895–899 (2003). Adachi, S., Nagae, K., and Matsuno, R.: Lipase-catalyzed condensation of erythritol and medium-chain fatty acids in acetonitrile with low water content. J. Mol. Catal. B: Enzym., 6, 21–27 (1999). Piao, J., Kobayashi, T., Adachi, S., Nakanishi, K., and
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(2003). 75. Arcos, J. A., Bernabé, M., and Otero, C.: Quantitative enzymatic production of 1,6-diacyl sorbitol esters. Biotechnol. Bioeng., 60, 53–60 (1998). 76. Humeau, C., Girardin, M., Coulon, D., and Miclo, A.: Synthesis of 6-O-palmitoyl L-ascorbic acid catalyzed by Candida antarctica lipase. Biotechnol. Lett., 17, 1091–1094 (1995). 77. Humeau, C., Girardin, M., Rovel, B., and Miclo, A.: Enzymatic synthesis of fatty acid ascorbyl esters. J. Mol. Catal. B: Enzym., 5, 19–23 (1998). 78. Humeau, C., Girardin, M., Rovel, B., and Miclo, A.: Effect of the thermodynamic water activity and the reaction medium hydrophobicity on the enzymatic synthesis of ascorbyl palmitate. J. Biotechnol., 63, 1–8 (1998). 79. Bradoo, S., Saxena, R. K., and Gupta, R.: High yields of ascorbyl palmitate by thermostable lipase-mediated esterification. J. Am. Oil Chem. Soc., 76, 1291–1295 (1999).
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80. Stamatis, H., Sereti, V., and Kolisis, F. N.: Studies on the enzymatic synthesis of lipophilic derivatives of natural antioxidants. J. Am. Oil Chem. Soc., 76, 1505–1510 (1999). 81. Yan, Y., Bornscheuer, U. T., and Schmid, R. D.: Lipasecatalyzed synthesis of vitamin C fatty acid esters. Biotechnol. Lett., 21, 1051–1054 (1999). 82. Watanabe, Y., Adachi, S., and Matsuno, R.: Condensation of L-ascorbic acid and medium-chain fatty acids by immobilized lipase in acetonitrile with low water content. Food Sci. Technol. Res., 5, 188–192 (1999). 83. Liu, K. J. and Shaw, J. F.: Lipase-catalyzed synthesis of kojic acid esters in organic solvents. J. Am. Oil Chem. Soc., 75, 1507–1511 (1998). 84. Chen, C. S., Liu, K. J., Lou, Y. H., and Shieh, C. J.: Optimisation of kojic acid monolaurate synthesis with lipase PS from Pseudomonas cepacia. J. Sci. Food Agric., 82, 601–605 (2002).
© 2005, The Society for Biotechnology, Japan
Synthesis of Esters by Immobilized-Lipase-Catalyzed Condensation Reaction of Sugars and Fatty Acids in Water-Miscible Organic Solvent SHUJI ADACHI1* AND TAKASHI KOBAYASHI2 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan1 and Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan2 Received 25 October 2004/Accepted 1 December 2004
A lipase-catalyzed condensation reaction in an organic solvent is a promising means of synthesizing esters. Reaction equilibrium constant, which is usually defined on the basis of reactant concentration, is an important parameter for estimating equilibrium yield. It is shown that the constant is markedly, affected by some factors, such as the hydration of a sugar substrate and the interaction of a reactant with a solvent. To reasonably design the reaction system or determine the reaction conditions, attention should be paid to these factors. From the viewpoint of kinetics, substrate selectivity for carboxylic acids also numerically correlates to the electrical and steric properties of these acids. Reactor systems for continuously producing esters through an immobilizedlipase-catalyzed condensation reaction are developed. [Key words: lipase, condensation, reaction equilibrium, ester, organic solvent]
under any conditions. However, only a few studies have shown the factors affecting such a constant for lipase-catalyzed condensation reaction in organic solvents. Most of investigations on lipase-catalyzed condensation reactions have been carried out using a small-scale batch reactor, although a continuous reaction is preferred for the large-scale production of esters. In this context, the factors affecting the reaction equilibrium constant will first be described, and the substrate se-
Esters are used in many foods, cosmetics, medicines and chemicals. Compared with conventional chemical syntheses, enzymatic preparation has some advantages, such as onestep synthesis without the protection and deprotection of polyols (1) and moderate reaction conditions. A lipase (EC 3.1.1.3) catalyzes a condensation reaction (reverse hydrolysis) to produce various esters, such as aliphatic alcohol esters (2–7), hydroxy fatty acid esters (8), polyesters (9, 10) and polyol esters (Table 1). Lipases are also widely used in various reactions, such as transesterification and alcoholysis, for the production of optically active compounds (11–17) and biodiesel fuel (18–20) as well as for the breakdown of fats and oil. The customization of lipases for practical use is extensively reviewed (21). In this study, the syntheses of esters by a lipase-catalyzed condensation reaction in watermiscible organic-solvent systems will be investigated. A hydrolase-catalyzed reaction in a conventional aqueous system thermodynamically favors hydrolysis. Because a lipase-catalyzed reaction is also such a case, a condensation reaction to produce an ester is carried out in a nonaqueous medium such as an organic solvent, a solvent-free system or an ionic liquid (22). Some lipases have catalytic activity even in the presence of small amounts of water (23). Reaction equilibrium constant is a crucial parameter in the prediction of the equilibrium yield of a desired product
TABLE 1. Synthesis of esters by lipase-catalyzed condensation reaction in organic solvent systems Alcohol substrate Glucose Glucose Fructose Mannose Galactose Methyl-a-D-glucoside Butyl-a-D-glucoside Octyl-a-D-glucoside Hydroxypropyl cellulose Glycerol Erythritol Xylitol Sorbitol Ribitol Ascorbic acid Kojic acid
* Corresponding author. e-mail: [email protected] phone: +81-(0)75-753-6286 fax: +81-(0)75-753-6285 87
Acid substrate Fatty acid Vinylacetic acid Fatty acid Fatty acid Fatty acid 1-Bromomyristic acid Fatty acid Cinnamic acid Fatty acid Fatty acid Fatty acid Fatty acid Fatty acid Fatty acid Fatty acid Fatty acid
References 1, 40, 41, 66, 67 45 36, 68 44, 59 60 69 70 71 72 37, 42, 73 56, 74 43 75 58 38, 64, 65, 76–82 52, 83, 84
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lectivity of a lipase for various carboxylic acids will follow. Finally, reactor systems for the continuous production of esters will be mentioned. The results obtained using only the lipase from Candida antarctica fraction B will be shown. CONDENSATION AND TRANSESTERIFICATION An ester can be synthesized through the lipase-catalyzed condensation of a carboxylic acid and an alcohol. Esters can also be produced by lipase-catalyzed transestrifications, which can be classified into alcoholysis, acidolysis and interesterification. In an organic solvent, alcoholysis uses methyl, ethyl and vinyl esters as substrates. Vinyl ester is most extensively used among esters because its by-product, vinyl alcohol, is almost irreversibly converted to acetaldehyde, thereby increasing the yield of the desired ester (24–30). Such conversion is generally higher in transesterification than in condensation (7, 31). However, the Schiff-base formation of acetaldehyde with lipase might present a problem (32). REACTION EQUILIBRIUM CONSTANT Definition of reaction equilibrium constant The condensation of an alcohol, A, and a carboxylic (or fatty) acid, F, results in the formation of a product (an ester), P, and a by-product (water), W. A+F
P+W
(1)
Reaction equilibrium constant, KC, is usually defined on the basis of the equilibrium concentrations, C, of the reactants because of its convenience in estimating the equilibrium yield of a desired product. KC = CPCW/(CACF)
(2)
However, reaction equilibrium constant, Ka, is thermodynamically defined on the basis of the activities, a, of the reactants by Ka = aPaW/(aAaF)
FIG. 1. Effects of initial water content on equilibrium yields of lauroyl glucose (closed circles), galactose (closed diamonds), mannose (closed squares) and fructose (closed triangles) and solubilities at 50°C of glucose (open circles), galactose (open diamonds), mannose (open squares) and fructose (open triangles) in acetonitrile (40).
this reaction system, the unreacted hexose remained in the solid state. To estimate the equilibrium concentration of each hexose, the solubility of the hexose in the solvent was measured at various water contents. The solubility could empirically be expressed as an exponential function of water content. The KC values for the synthesis of the lauroyl hexoses were evaluated. KC significantly depended on the type of hexose even though the bond formed by a condensation reaction is common for every hexose. As a possible reason for the dependence, the hydration of hexose was considered to affect equilibrium conversion. The hexose that is more strongly hydrated further decreased the water activity in the system to shift the equilibrium toward synthesis. The dynamic hydration number of hexose (34, 35) was selected as a measure of the extent of hydration. As shown in Fig. 2, there was a positive correlation between KC and the dynamic hy-
(3)
Ka is intrinsic under specific temperature and pressure, while KC is apparent because it is affected by environmental conditions. Although there is an attempt to estimate a with the UNIFAC (33), it is either impossible or extremely difficult to evaluate all the a values of the reactants. This is also a reason for the convenient use of KC. Ka and KC are related to each other through the activity coefficients, g, of the reactants by gA gF KC = -----------K (4) gP gW a Effects of water on reaction equilibrium Hydration of a saccharide Figure 1 shows the equilibrium conversions of lauroyl glucose, galactose, mannose and fructose in acetonitrile with various initial water contents. The equilibrium conversion was low at a high water content in every solvent, though this tendency is reasonable because water is one of the products of condensation. The term conversion indicates the molar ratio of a desired product to a limiting substrate, hexose (generally, alcohol substrate). In
FIG. 2. Correlations of the apparent equilibrium constant KC for lauroyl hexose synthesis in acetonitrile with the dynamic hydration number of hexoses (closed squares) (40), and of KC for lauroyl mannose synthesis with relative dielectric constant of various solvents (open squares) (44). 2M2P, 2-Methyl-2-propanol; 2M2B, 2-methyl2-butanol.
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FIG. 3. Equilibrium conversion for lauroyl mannose synthesis in 2-methyl-2-propanol (circles) and 2-methyl-2-butanol (triangles) at various amounts of molecular sieve 3A (39). Inset: adsorption isotherms at 50°C of water onto molecular sieve 3A in the solvents.
dration number, indicating that water activity plays an important role in the condensation reaction in organic solvents. Condensation in the presence of desiccant Water is formed by a condensation reaction. Its removal from a reaction system is effective in shifting the reaction toward ester formation. For this reason, the addition of a desiccant (i.e., a molecular sieves [1, 31, 36–38] or silica gel [5, 10]) to a reaction system has often been adopted. However, there is as yet no criterion for the amount of desiccant to be added to achieve the desired conversion. A method of predicting equilibrium conversion for monoacyl hexose synthesis by a lipase-catalyzed condensation reaction in a water-miscible solvent in the presence of a molecular sieve has been proposed. The method is based on the adsorption isotherm of water onto molecular sieves, KC, the solubility of the hexose in the solvent, and the mass balance of water. Figure 3 shows the apparent equilibrium conversions of mannose and lauric acid to lauroyl mannose by an immobilized lipase in 2-methyl-2-propanol and 2-methyl-2-butanol in the presence of various amounts of molecular sieve 3A (39). The conversion was high in the presence of large amounts of the molecular sieve. The solid curves in Fig. 3 were calculated by the proposed method and expressed well the experimental results. The inset of Fig. 3 shows the adsorption isotherms at 50°C of water onto molecular sieve 3A in the solvents. The isotherms are expressed by the Langmuir equation. The molecular sieve acts as a catalyst as well as an adsorbent. There are some cases in which undesirable reactions, such as the degradation of unstable substrates and diester formation, proceed (10, 36, 38). Solvent effects on the reaction equilibrium Both water-miscible and -immiscible organic solvents have been used as reaction media for lipase-catalyzed condensation reactions. The use of water-miscible solvents, such as acetone, acetonitrile and tertiary alcohols, has the advantage that hydrophilic substrates, such as saccharides, are solubilized to some extent without the addition of solubilizing reagents to facilitate the esterification of the substrates (1, 40– 43). However, a water-miscible solvent removes water from
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a lipase molecule, which is essential for catalytic activity; the removal might bring about the deactivation of the lipase. A condensation reaction is usually performed in a pure solvent with a low water content. Even though the solvent provides only the field of the reaction, there are some cases in which the type of solvent affects equilibrium conversion. Changing the solvent from hexane to tertiary alcohols suppresses diester (triester) formation preceding monoester formation for the synthesis of oleoyl xylitols (43). The difference in equilibrium conversion was discussed on the basis of the polarity of the solvent. Lauroyl mannose (44) and vinylacetyl glucose (45) were synthesized by a lipase-catalyzed condensation reactions in various water-miscible solvents. The KC values for lauroyl mannose formation differed by about two orders of magnitude among the solvents. The KC values were the highest in acetonitrile, intermediate in acetone, and low in 2-methyl-2-propanol and in 2-methyl2-butanol. A log P value, where P is the partition coefficient between 1-octanol and water phases, is most frequently used in characterizing a solvent (4, 46). An empirical DimrothReichardt parameter, ET(30), expressing the polarity of a solvent (47) is also used in assessing the enzyme stability in organic solvents (48). The correlations of KC with log P, ET(30) and the relative dielectric constant of the solvent were examined. KC (ln KC) correlated best with the relative dielectric constant as shown in Fig. 2 (44). However, the dielectric constant was not applicable in analyzing the formation of butyl decanoate (6). Therefore, the correlation obtained for lauroyl mannose synthesis seemed to be apparent, as will be discussed later. A pure organic solvent with inherent properties is usually used as a reaction medium. The mixing of two different solvents can adjust the values of the properties between their inherent values, and allows us to examine the solvent effect on reaction equilibrium in detail. Oleoyl xylitols were synthesized in mixed solvents and the equilibrium conversion to a diester was found to increase with decreasing ET(30) (43). KC for butyl decanoate synthesis was estimated in mix-
FIG. 4. Apparent equilibrium constant for butyl decanoate synthesis in mixtures of a tertiary alcohol and a nitrile at various ratios. The open and closed symbols represent the mixtures of 2-methyl-2-propanol and 2-methyl-2-butanol, respectively, with acetonitrile (diamonds), propionitrile (squares) and butyronitrile (triangles) (6).
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TABLE 2. Carboxylic acids used for examination of substrate selectivity Non-conjugated Conjugated
Substrate no. 1 2 3 7 8
Straight chain Propionic acid Butyric acid Vinylacetic acid Acrylic acid Crotonic acid
tures of a nitrile and a tertiary alcohol at various ratios (6). As shown in Fig. 4, KC increased with the fraction of nitrile. The alkyl chain length of the nitrile or tertiary alcohol only slightly affected KC. These results suggest that the reaction equilibrium can be, to a certain degree, controlled using a mixture of a nitrile and a tertiary alcohol as the reaction medium, and that the polar region of a solvent molecule markedly affects KC. The infrared (IR) spectra of decanoic acid and butyl decanoate were measured in mixtures of a nitrile and a tertiary alcohol. The IR absorption peak of C = O for decanoic acid shifted to a higher wave number at a higher fraction of acetonitrile in the mixture with 2-methyl-2-propanol. The change in wave number for butyl decanoate was smaller than that for decanoic acid. The KC estimated for the mixture of acetonitrile and 2-methyl-2-propanol, on a semi-logarithmic scale, could linearly correlate to wave number at the peak of the C= O double bond of decanoic acid. These results indicate that decanoic acid (fatty acid) interacts more strongly with 2-methyl-2-propanol (a tertiary alcohol) than with butyl decanoate (ester).
Substrate no. 4 5 6 9 10
Branched chain Isobutyric acid Isovaleric acid Cyclohexanecarboxylic acid Methacrylic acid Benzoic acid
also, on a semi-logarithmic scale, plotted against the projection area. The plots for the conjugated acids with straight and branched chains form a straight line, and those for the nonconjugated acids form another straight line. The two lines are almost parallel. These results roughly indicate that the presence of a conjugation or a branched chain independently lowers Vmax/Km by one order; presence of both decreases Vmax/Km by two orders. REACTORS FOR CONTINUOUS SYNTHESIS OF ESTERS A batch reactor is generally used for the lipase-catalyzed
SUBSTRATE SELECTIVITY FOR CONDENSATION REACTION There are some cases in which a condensation reaction proceeds rather slowly, e.g., the synthesis of ferulic acid esters (7). Although the electrical and steric effects of substrates on reaction rate were reported (49, 50), no attempt was made to numerically correlate the effects with substrate selectivity. The maximum reaction rate Vmax and the Michaelis constant Km were evaluated for the condensation reactions of various short-chain carboxylic acids with p-methoxyphenethyl alcohol (51). The carboxylic acids used as the substrate can be classified into four groups by the presence of branched chains and conjugations in their molecular structures (Table 2). The steric and electrical properties of the substrate molecules were estimated from the optimized structure of a carboxylic acid molecule and the electron density of a carboxylic carbon using the molecular orbital calculation software MOPAC 2000. Figure 5 shows the relationship between Vmax/Km and the electron density of a carboxyl carbon of an acid. The Vmax/Km values of the nonconjugated acids were about 10 times higher than those of the conjugated ones; those of the acids with straight chains were also about 10 times higher than those of the acids with branched chains. Figure 6 shows the relationship between the projection area of the non-carboxylic region of an acid and the Km of the acid. The Km is linearly related to the projection area, indicating that a more bulky acid exhibits a greater steric hindrance to the formation of the enzyme-substrate complex. The Vmax/Km is
FIG. 5. Relationship between log Vmax/Km of acid and electron density of carboxyl carbon of acid (51). Vmax/Km is expressed in min–1. Labels in the graph correspond to the substrate number in Table 2.
FIG. 6. Effects of projection area of non-carboxylic region of acid molecule on log Vmax/Km and Km of acid (51). Vmax/Km is expressed in min–1. Labels in the graph correspond to the substrate number in Table 2.
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FIG. 7. Continuous syntheses of oleoyl (squares), linoleoyl (reverse triangles), decanoyl (circles), lauroyl (diamonds) and myristoyl (triangles) L-ascorbates using reactor system illustrated in graph (55). 1, Feed reservoir; 2, pump; 3, preheating coil; 4, column packed with ascorbic acid powders; 5, column packed with immobilized lipase; 6, thermoregulated chamber; 7, effluent reservoir.
synthesis of esters, although a continuous reactor would be preferred for their large-scale production. Continuous reactors are typically classified into a continuous stirred tank reactor (CSTR) and a plug flow reactor (PFR) on the basis of the type of liquid mixing in the reactor. Because the solubility of an alcohol substrate, such as saccharide, in an organic solvent is low, the substrate remains undissolved in the solvent when an excess amount of the substrate is added. Therefore, CSTR seems to be a candidate system for the continuous production of an ester through a lipase-catalyzed condensation reaction in a solvent. We realized the continuous or semicontinuous syntheses of fatty acid esters of kojic acid (52), maltose (53) and ascorbic acid (54) using CSTR. The solubility of an alcohol substrate in an organic solvent generally ranges from 1 to 50 mmol/l. For a product, if we assume that its conversion is 0.7 and its molecular mass is 300, its concentration range 0.2–10 g/l. This concentration is not high, but is not extremely low either. Based on such a hypothetical calculation, a PFR system for the continuous production of an ester was proposed, as shown in the inset of Fig. 7. Powders of an alcohol substrate were packed into a column, immobilized-lipase particles were packed into another column, and the columns were then connected in series. A fatty acid solution dissolved in an organic solvent was fed to the alcohol substrate column to dissolve the substrate to a concentration close to saturation, and then pumped to the lipase column to produce an ester. Figure 7 shows the continuous synthesis of various acyl ascorbates using such a reactor system (55). Each acyl ascorbate was produced for 2 d. The system could be stably operated for at least 10 d, and the product concentrations in the effluent were in the range of 14 to 17 mmol/l. These product concentrations corresponded to the productivity of 1.6 to 1.9 kg/l-reactor/d, depending on the molecular mass of the product.
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FIG. 8. Oxidation of a-linolenic (open reverse triangles), eicosapentaenoic (open circles) and docosahexaenoic (open diamond) acids, and a-linolenoyl (closed reverse triangles), eicosapentaenoyl (closed circles) and docosahexaenoyl (closed diamonds) L-ascorbates at 65°C and almost 0% relative humidity (64, 65).
The PFR system was successfully applied to the continuous synthesis of fatty acid esters of erythritol (56, 57), various sugar alcohols (58) and hexoses (59, 60). The properties of the esters synthesized through a lipasecatalyzed condensation reaction are described in terms of their applications. As an example, the properties of saturated or unsaturated acyl ascorbate will briefly be described. The radical scavenging activity of ascorbic acid is not affected by the introduction of an acyl group to the hydroxyl group at the C-6 position of the ascorbic acid (54). Although ascorbic acid is insoluble in soybean oil, the acylation of ascorbic acid largely improves its solubility in the oil (54). Saturated acyl ascorbates were surface-active and their critical micelle concentrations were measured (61). The acyl ascorbates can be used in the microencapsulation of an unsaturated fatty acid with a polysaccharide by spray drying and can effectively suppress the oxidation of the encapsulated fatty acid (62). The ascorbates were also effective for suppressing the oxidation of the membrane of intestinal epithelial cells (63). Polyunsaturated fatty acids are prone to oxidation. Their esters with ascorbic acid are very resistant to oxidation. Figure 8 shows the oxidation processes of the unmodified a-linolenic, eicosapentaenoic and docosahexaenoic acids and the acyl moiety of their ascorbates at 65°C and almost 0% relative humidity (64, 65). The unmodified fatty acids were completely oxidized within 2 h, while all the unsaturated acyl moieties of the ascorbates remained unoxidized in that period. It was also shown that the addition of a saturated acyl ascorbate to an unsaturated fatty acid significantly suppresses the oxidation of the unsaturated fatty acid (65). REFERENCES 1. Arcos, J. A., Bernabé, M., and Otero, C.: Quantitative enzymatic production of 6-O-acylglucose esters. Biotechnol.
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