Ester Synthesis From Trimethylammonium Alcohols in Dry Organic Media Catalyzed by Immobilized Candida antarctica Lipase B Pedro Lozano, Mirta Daz,* Teresa de Diego, Jose´ L. Iborra Departamento de Bioquı´mica y Biologı´a Molecular B e Inmunologı´a, Facultad de Quı´mica, Universidad de Murcia, P.O. Box 4021, E-30100 Murcia, Espan˜a; telephone: 34-968-36-7398; fax: 34-968-36-4148; e-mail:
[email protected] Received 16 May 2002; accepted 8 October 2002 DOI: 10.1002/bit.10580
Abstract: Twenty-one different organic solvents were assayed as possible reaction media for the synthesis of butyryl esters from trimethylammonium alcohols in dry conditions catalyzed by immobilized Candida antarctica lipase B. The reactions were carried out following a transesterification kinetic approach, using choline and Lcarnitine as primary and secondary trimethylammonium alcohols, respectively, and vinyl butyrate as acyl donor. The synthetic activity of the enzyme was strictly dependent on the water content, the position of the hydroxyl group in the trimethylammonium molecule, and the Log P parameter of the assayed solvent. Anhydrous conditions and a high excess of vinyl butyrate over L-carnitine were necessary to synthesize butyryl-L-carnitine. The synthetic reaction rates of butyryl choline were practically 100-fold those of butyryl-L-carnitine with all the assayed solvents. In both cases, the synthetic activity of the enzyme was dependent on the hydrophobicity of the solvent, with the optimal reaction media showing a Log P parameter of between −0.5 and 0.5. In all cases, 2-methyl-2-propanol and 2-methyl-2-butanol were shown to be the best solvents for both their high synthetic activity and negligible loss of enzyme activity after 6 days. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 352–358, 2003.
Keywords: lipase; trimethylammonium ester; butyryl-Lcarnitine; butyryl choline; ester synthesis
INTRODUCTION The technological usefulness of enzymes can be greatly enhanced if they are used in organic media rather than in their natural aqueous reaction media. In this way, enzymes in nonconventional media may exhibit new catalytic properties, such as synthetic activity (by hydrolytic enzymes), stereo- and regioselectivity for substrates, and enhanced stability (Klibanov, 2001). Among enzymes, lipases have been used successfully as a catalyst for the synthesis of esters, Correspondence to: J. L. Iborra *Permanent address: Departamento de Quı´mica. Facultad de Ciencias Exactas, Universidad Nacional de Salta, Salta, Repu´blica Argentina Contract grant sponsor: CICYT Contract grant number: BIO99-0492-C02-01
© 2003 Wiley Periodicals, Inc.
both on a small scale and on an industrial scale (Balc˜o et al., 1996; Jaeger and Reetz, 1998). Recently, research has been focusing on amphiphilic molecules, for example, fatty acid sugar esters (Arcos et al., 1998) and dihydroxyacetone fatty acid esters (Virto et al., 2000) for use as nonionic surfactants, esters of ␣-hydroxyacids as humectants (Torres and Otero, 1999; Torres et al., 1999), and phospholipids (Virto et al., 1999; Virto and Adlercreutz, 2000) for their important food and pharmaceutical applications. Lipase-catalyzed synthesis of these compounds requires nearly anhydrous conditions and the use of nonpolar solvents as reaction media to avoid the stripping of essential water molecules from the enzyme structure. However, polar acyl-acceptors exhibit limited solubility in common “enzyme-friendly” organic solvents (e.g., hexane). Several strategies have been used to overcome this problem, including the complexation of polar substrates (Castillo et al., 1994), the adsorption of polar substrates on inert silica-gel (Castillo et al., 1997), as well as the use of hydrophilic solvents and/or free-solvent systems containing partially soluble polar substrates (Ljunger et al., 1994). The use of polar solvents or free-solvent systems combined with an efficient removal of water from the media has proved to be an adequate strategy for enzymatic ester synthesis (Torres et al., 1999; Virto et al., 1999). Both direct esterification and transesterification approaches have been assayed in the lipase-catalyzed synthesis of amphiphilic esters. Among the numerous acyl-donors employed during transesterification, vinyl esters are the most popular, because the vinyl alcohol formed during the process tautomerizes to low-boiling-point acetaldehyde, thus shifting the equilibrium toward ester formation (Virto and Adlercreutz, 2000). Choline and L-carnitine [R-(-)-3-hydroxy-4-N,N,N-trimethylaminobutyrate] are ubiquitous trimethylammonium alcohols, which play different roles in living cells. In addition to the classical functions of these alcohols, many pharmacological applications have been described for their
derivatives. For example, long-chain esters of choline and L-carnitine exhibit activity against bacteria, yeasts, and fungi (Ahlstroem et al., 1995; Calvani et al., 1998), whereas short-chain esters of L-carnitine are used for cardiovascular treatment (Arsenian, 1997), dermatoses (Cavazza and Cavazza, 1994), and Alzheimer’s disease (Calvani and Carta, 1996). In all cases, both choline and L-carnitine esters are obtained by chemical synthetic processes. This study describes, for the first time, the enzymatic synthesis of butyryl esters of both choline and L-carnitine in dry conditions, using 21 different organic solvents as organic reaction media. Novozyme 435 (Candida antartica lipase B immobilized on acrylic macroporous resin) was used as catalyst, and synthetic reactions were carried out by transesterification, using vinyl butyrate as acyl-donor.
medium was incubated with gentle shaking overnight at 40°C to reach solubility equilibrium. The reaction was started by adding 20 mg of immobilized enzyme and run at 40°C with shaking. At regular time intervals, 50-mL aliquots were extracted, centrifuged for 3 min at 13,000g, and the resulting solutions were analyzed by high-performance liquid chromatography (HPLC). At the end of the assay, the overall solid fraction was recovered and divided in two parts. From the first part, the immobilized enzyme particles were recovered and then used to quantify the residual esterase activity. The second part was suspended in dry ethanol, then mixed for 5 min, and finally centrifuged for 3 min at 13,000g. A 50-mL aliquot was analyzed to quantify the possible precipitated product by HPLC. Experiments were carried out in duplicate.
MATERIALS AND METHODS
HPLC Analysis
Chemicals and Enzyme
Substrate and product concentrations were determined by HPLC (Shimadzu LC10) using a LiChrocart RP18 column (12.5-cm length and 4-mm internal diameter, 5-mm particle size, and 10-nm pore size). Samples (50 mL) containing choline derivatives were eluted in a linear gradient (Phase A: acetonitrile 30% with 10 mM SDS and 15 mM phosphoric acid; Phase B: acetonitrile 60% with 10 mM SDS and 15 mM phosphoric acid) at 1.5 mL min−1, using 1 mM acetanilide in ethanol, as internal standard. Elution profiles were monitored at 210 nm, and showed a detection limit of 0.25 mM. Samples containing L-carnitine and L-butyrylcarnitine were chemically modified to a chromophore for UV detection, using a modification of the method described by Kagawa et al. (1999). Two hundred microliters of appropriately diluted sample in ethanol were added to a screwcapped vial with a Teflon seal, containing 0.2 mL of 50 mM ethyl chloroformate in chloroform, 0.2 mL of 50 mM triethylamine in chloroform, and 0.2 mL of 20 mM ␣-naphthylamine in ethanol. The mixture was incubated at 40°C for 30 min, reaching the full chromophoric modification. Then, 20 L of 0.1 M HCl was added to avoid hydrolysis of L-butrylcarnitine to L-carnitine by TEA. The chomophoric product mixture was stable for 2 days at 4°C. Finally, 50 L of internal standard solution (100 mM acetanilide in ethanol) was added. The final injection sample (50 mL), containing substrate and product derivatives at the appropriate dilution in ethanol, was eluted in a linear gradient (Phase A: acetonitrile 10% in 20 mM TEA/phosphoric acid buffer solution, pH 2.5; Phase B: acetonitrile 80% in 20 mM TEA/ phosphoric acid buffer solution, pH 2.5) at 1 mL min−1, and monitored at 280 nm. Standard calibration was carried out using either L-carnitine, L-butyrylcarnitine, or butyric acid standard solutions (from 0.01 to 1 mM) in ethanol. The resulting straight-line equations were respectively as follows: y ⳱ 0.657x + 1.12 ⭈ 10−4 (r2 ⳱ 0.998), y ⳱ 0.754x + 2.51 ⭈ 10−4 (r2 ⳱ 0.997), and y ⳱ 0.546x + 3.56 ⭈ 10−4 (r2 ⳱ 0.995), where y is the ratio between the integrated
Candida actarctica lipase B immobilized on a macroporous resin (Novozyme 435) was a gift from Novo-Nordisk A/S (Bagsværd, Denmark). L-carnitine, choline, butyryl choline, and ␣-naphthylamine were obtained from Sigma Co. (St. Louis, MO). Ethyl chloroformate, vinyl butyrate, triethylamine, sodium dodecylsulfate (SDS), acetanilide, molecular sieve UOP Type 4 (pore diameter 4 Å), and butyric acid were from Fluka. All of the remaining reagents and solvents were of analytical grade. Drying of Chemicals and Water Content Analysis Water was removed from the solvents and vinyl esters by adding molecular sieves (0.1 g/mL), shaking the resulting mixture for 24 h at room temperature, and finally storing them in the presence of the adsorbent. Both choline and L-carnitine were dried as follows: 0.5 g of trimethylammonium alcohol were added to 25 mL of dry acetone, and the resulting mixture was stirred for 1 h at room temperature. Then, the remaining solid was recovered by filtration and dried under nitrogen. After the drying process, the remaining water content, as determined by the Karl-Fisher (MKS 210 Kyoto Electronics) method, of all the components of the reaction media ranged from 0.02% to 0.06% (w/w) for solvents, whereas choline and L-carnitine showed 0.5% and 0.95% (w/w) water contents, respectively. Novozyme was used without any drying process, showing a 3.5% (w/w) water content. Synthetic Reactions One hundred micromoles of choline or L-carnitine and 100 mg of molecular sieves were placed in a screw-capped vial with a Teflon seal. Then, 1 mL of an appropriate mixture of vinyl ester and solvent was added, and the resulting reaction
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areas of the compound and the internal standard, and x is the ratio between the concentrations of the compound and the internal standard. The detection limit exhibited by this method was 0.02 mM. The L-butyrylcarnitine used as standard for HPLC was synthesized according to the method of Bohmer and Bremer (1968). One unit of synthetic activity was defined as the amount of enzyme that produced 1 mol of product per minute. Assay of Esterase Activity The esterase activity of Novozyme (fresh and recovered from organic media) was assayed tritrimetrically, using a video-titrator VIT-90 equipped with an autoburette (ABU 91) and a sample station (SAM 90, Radiometer, Copenhagen), under the following conditions: a solution containing 0.1 M KCl, 1% (w/v) sodium deoxycholate, and 6% (v/v) ethyl hexanoate was sonicated over 3 min and its pH adjusted to 7.0 with 10 mM NaOH. Ten milliliters of this solution was incubated at 40°C and the reaction was started by the addition of 20 mg of Novozyme. The pH was maintained constant at 7.0 by continuous addition of 10 mM NaOH as titrant. One unit of activity was defined as the amount of enzyme that hydrolyzes 1 mol of ethyl hexanoate per minute. RESULTS AND DISCUSSION Effect of Solubility of Trimethylammonium Alcohols In lipase-catalyzed reactions, a covalently linked acylenzyme intermediate is formed (see Fig. 1A). Nucleophilic attack of this intermediate by water resulted in ester hydrolysis, although the presence of another nucleophile (e.g., R⬘-OH) may involve the formation of the transesterification product. This synthetic pathway can be regarded as a kinetically controlled process, where the rapid accumulation of the acyl-enzyme intermediate and preferential nucleophilic attack, determined at kT[R⬘-OH] >> kH[H2O], are essential. The first condition is enhanced by the use of activated acid acyl-donors as vinyl esters, because the vinyl alcohol released in the degradation of the vinyl ester tautomerizes to acetaldehyde, which cannot act as a substrate for the enzyme (Degueil-Castaing et al., 1987; Virto and Adlercreutz, 2000). The second condition may arise from using both reaction media with very low water content and a high nucleophile (R⬘-OH)/acyl donor concentration ratio. However, the use of quaternary ammonium alcohols (e.g., choline or L-carnitine; Fig. 1B) as acyl-acceptors would greatly limit the extension of the synthetic pathway due to their poor solubility in many of the organic solvents used as reaction media (see Table I). Thus, an increase in the Log P parameter of the solvent reduces the solubility of both substances. The solubilization of choline and L-carnitine molecules involves bond–dipole interactions with solvent mol-
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Figure 1. (A) Synthesis of esters from vinyl ester and alcohols catalyzed by lipase. (B) Structure of choline and L-carnitine.
ecules due to the charged nitrogen atom they contain, both compounds being nonsoluble in hydrophobic solvents. In this way, Calvani et al. (1998) described a linear correlation between the capacity factors (k⬘) in a C18 column and the calculated Log P (CLOGP) of 34 different quaternary ammonium L-carnitine esters, which contained long-chain alkyl substituents. The large influence of the charged nitrogen on the polar properties of these compounds is demonstrated by the low differences in the experimental k⬘ parameter, suggesting that all the substances exhibit similar amphipathicity, with no dependence on the nature of the alkyl substituent. In spite of the low solubility of choline and L-carnitine in dry organic solvents, the synthesis of butyryl ester derivatives from these quaternary ammonium compounds catalyzed by Novozyme 435 was assayed in 21 different organic solvents. The synthetic yields obtained with each solvent at 40°C are also shown in Table I. However, it should first be pointed out that when the reactions proceeded in the absence of molecular sieves no synthetic products were obtained, so that inclusion of this dehydrating agent in the reaction media was absolutely necessary. Also, synthetic products were not detected in the insoluble fraction of the reaction media, suggesting that a solubilization of trimethylammonium substrates occurred during the process for solvents with limited solubilization ability. As can be seen, the synthetic yield obtained was strongly dependent on the nature of the assayed solvent for both butyryl trimethylammonium esters. 2-Methyl-2-butanol was shown to be the best solvent for the lipase-catalyzed synthesis of butyryl choline (19.0 U/g initial synthetic activity, 91% synthetic product yield at 24 h), whereas, in the case of butyryl-L-carnitine, the best results were obtained with acetonitrile (0.17 U/g initial synthetic activity, 66.4% synthetic product yield at 6 days). In addition, neither synthetic product was detected in reactions carried out in any of the water-immiscible solvents. A comparison of the data included in Table I shows how the solubility of these trimethylammonium alcohols was not the only factor, because the best solvents for dissolving the
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Table I. Solubility of choline and L-carnitine, determined by HPLC from saturated solution, in different dry organic solvents after 24 h of magnetic stirring at 40°C. Solubility
Yield
Log P
Choline (M)
L-carnitine
Solvent
(mM)
BuChol at 24 hours (%)
BuCar at 6 days (%)
Tetra(ethylene-glycol) dimethyl ether Formamide Di(ethylene-glycol) dimethyl ether Dioxane N,N-dimethylformamide N-methylformamide Acetonitrile Di(ethylene-glycol) diethyl ether Acetone Propionitrile 2-Methyl-2-propanol Tetrahydrofurane Diethylether 2-Methyl-2-butanol 3-Methyl-2-pentanol Benzene Toluene Tetrachloromethane n-Hexane n-Heptane n-Octane
−2.47 −1.61 −1.30 −1.10 −1.01 −0.97 −0.33 −0.27 −0.23 0.16 0.35 0.49 0.85 0.89 1.71 2.00 2.50 3.00 3.50 4.00 4.50
NA 2.5 NA NA 0.5 1.7 17 × 10−3 NA NA NA 25 × 10−3 NA NA 12 × 10−3 NA ND ND ND ND ND ND
0.2 3.0 2.0 ND 2.0 1.0 2.6 1.0 1.2 0.7 10.5 ND ND 10.5 2.1 ND ND ND ND ND ND
NA 29.7 NA NA 18.0 32.0 70.0 NA NA NA 78.0 NA NA 91.0 NA 0.0 0.0 0.0 0.0 0.0 0.0
8.0 3.7 20.4 0.0 5.2 19.7 66.4 5.4 55.8 48.5 45.2 0.0 0.0 55.9 7.0 0.0 0.0 0.0 0.0 0.0 0.0
Synthetic yield of butyryl choline (BuChol) and butyryl-L-carnitine (BuCar), with respect to the initial choline and L-carnitine amount (100 micromol), produced by immobilized CALB (Novozyme 435) in different dry organic solvents at 40°C. Initial vinyl butyrate concentration was 20% (v/v). ND, not detected; NA, not assayed.
substrates were not the best reaction media. For example, formamide was able to dissolve 2.5 M choline, but the initial synthetic activity of the enzyme in this solvent (0.9 U/g) was 21-fold lower than that in 2-methyl-2-butanol. These findings could be explained by both the solvent–water and solvent–enzyme interactions that took place. In all cases (Table II), the water content of the reaction media was very low, and the ability of the solvents to sequester essential water molecules from the microenvironment of the immobilized enzyme in these anhydrous media may lead to enzyme deactivation by water-stripping (Klibanov, 2001; Levitsky et al., 2000). Table II shows the initial activity of the immobilized enzyme for butyryl-L-carnitine synthesis in the best solvents at 40°C, as well as the residual esterase activity of the recovered immobilized enzyme from these media after 6 days of operation. As can be seen, an increase in solvent polarity reduced the initial synthetic activity and increased activity loss. The synthetic activity of lipase produced good yields in those polar solvents, which showed an ability to dissolve a critical amount of choline and Lcarnitine (i.e., 2-methyl-2-butanol), with negligible deactivation effects. Kinetic Characteristics of Synthesis of Trimethylammonium Esters The solvent 2-methyl-2-butanol was chosen to analyze the kinetic characteristics of the process. Figure 2 shows the
time-course of both the hydrolytic (butyric acid) and synthetic (butyryl choline or butyryl-L-carnitine) products obtained in 2-methyl-2-butanol by the Novozyme 435 action. For both trimethylammonium alcohols, the hydrolytic activity of the enzyme was clearly higher than the synthetic activity, even when all the components of the reaction media were previously dried, and a large quantity of molecular sieves (100 mg/mL) was included. Butyryl choline was syn-
Table II. Initial water content and initial synthetic activity of immobilized CALB for butyryl-L-carnitine synthesis in different organic solvents at 40°C.
Solvent
Water content (mM)
Initial synthetic activity (U/g)
Residual esterase activity (%)
Di(ethylene-glycol) dimethyl ether N-methylformamide Acetonitrile Acetone 2-Methyl-2-propanol 2-Methyl-2-butanol
47.2 45.5 29.4 66.5 79.4 119.3
0.06 0.06 0.16 0.11 0.12 0.14
48.5 49.9 59.8 66.7 98.7 99.5
All synthetic reaction media contained 1.56 M (20% v/v) dry vinyl butyrate in dry solvent, 100 mol in total of dry L-carnitine, 100 mg of molecular sieves, and 20 mg of Novozyme 435. Residual esterase activity (see “Materials and Methods”) was determined using the recovered immobilized enzyme from the different reaction media after 6 days. a Initial esterase activity 22.1 U/g.
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Figure 2. Time-courses of reaction products (䊉, butyric acid; 䉱, butyryl choline; 䊏, butyryl-L-carnitine) for immobilized CALB-catalyzed ester synthesis from choline (A) and L-carnitine (B) in 2-methyl-2-butanol at 40°C. Both reaction media contained 1.56 M (20% [v/v]) dry vinyl butyrate, 100 mmol (total) of dry choline or L-carnitine, 100 mg of molecular sieves, and 20 mg of Novozyme 435.
thesized much faster than butyryl-L-carnitine, with a 60% butyryl choline yield being obtained after 2 h (Fig. 2A), whereas reaching the same yield of butyryl-L-carnitine took 6 days (Fig. 2B). These results were the consequence of the low nucleophilic power of L-carnitine than of choline, due to the secondary position of the hydroxyl group in the former and the primary position in the latter (Fig. 1B). Taking into account the low solubility of these trimethylammonium alcohols, the hydrolytic activity seems to be a kinetic way of controlling free water molecules for two reasons. First, in both cases, this activity was stopped when only 6% of the initial vinyl butyrate concentration (1.56 M) had been consumed. As the hydrolytic activity consumed free water molecules, it can also be seen how the final butyric acid concentration was about 80% of the initial water content (see Table II), whereas the remaining water content of the medium may correspond to non-free water molecules. Second, because L-carnitine has a lower nucleophilic power than choline, the enzymatic synthesis of butyryl-L-carnitine proceeds at a lower water concentration than in the case of butyryl choline (see Fig. 1A). Hence, no butyryl-L-carnitine was synthesized during the first day of the reaction, whereas the hydrolytic product was obtained very rapidly. After the hydrolytic activity had reduced the water concentration to a critical level (approx. 50 mM), butyryl-L-carnitine synhesis was started by the enzyme. Furthermore, it is necessary to point out that the concentration of both trimethylammonium substrates was constant during the firstorder step of the synthetic reaction, indicating that the solid fraction of the substrate was a nucleophile reservoir. These results clearly demonstrate the kinetic control of the water content of the synthetic activity exhibited by the enzyme. Ljunger et al. (1994) reported the C. antarctica lipasecatalyzed synthesis of octanoylglucose from insoluble glu-
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cose and octanoic acid in acetonitrile. In this case, wherein the glucose was not fully soluble in the solvent, the enzyme was also able to catalyze the synthetic products at high acid concentration. Figure 3 shows the influence of the vinyl butyrate (acyldonor) concentration on the ability of Novozyme 435 to catalyze the synthesis of butyryl choline and butyryl-Lcarnitine in 2-methyl-2-butanol in the presence of the same trimethylammonium alcohol (100 mmol) and molecular sieve (100 mg/mL) content. As can be seen, in both cases, the synthetic activity was clearly dependent on the concentration of vinyl butyrate, with the critical concentration being 0.3 M. This coincides with the kinetic control of the water concentration in the reaction media, wherein a decrease in water content as a result of the hydrolytic activity of the enzyme allows for the expression of synthetic activity. In the case of choline ester (Fig. 3A), the synthetic activity was enhanced exponentially to its maximum level (16 U/g), which was observed at an acyl-donor concentration of 1 M. However, in the case of butyryl-L-carnitine synthesis (Fig. 3B), the maximum activity (0.15 U/g) was reached at an acyl-donor concentration 1.56 M. At above this concentration, there was a gradual reduction in synthetic activity until no activity was seen at 8 M (100% [v/v]) vinyl butyrate. In this case, vinyl butyrate plays a double role, as substrate and solvent, and its low polarity (Log P ⳱ 1.71) was not able to dissolve L-carnitine. In addition, it can be seen how the ability of the enzyme to synthesize butyryl choline was 100-fold higher than in the case of butyryl-L-carnitine. As mentioned earlier, secondary alcohols are poorer nucleophiles than primary alcohols. In addition, from the lipase-catalyzed esterification of the secondary hydroxyl group of lactic acid by decanoic acid, From et al. (1997) observed that the steric hindrance produced by the presence of the carboxyl group of the nucleophile is important in determining the decrease in enzymatic synthetic activity.
Figure 3. Effect of vinyl butyrate concentration on the ability of Novozyme 435 to catalyze the synthesis of butyryl choline (A, 䊉) and butyrylL-carnitine (B, 䊏) in 2-methyl-2-butanol at 40°C. See Figure 2 for other reaction conditions.
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Influence of Solvent Hydrophobicity on Trimethylammonium Ester Synthesis To ascertain the influence of the polar characteristics of the solvent used as reaction medium, the ability of Novozyme 435 to synthesize both trimethylammonium esters was assayed with 21 different solvents (listed in Table II) in dry conditions and the optimum vinyl butyrate concentration as determined earlier. Figure 4 shows the evolution of both the initial butyryl choline and butyryl-L-carnitine synthetic activity as a function of Log P. As can be seen, the immobilized enzyme showed an increase in both synthetic activities when Log P decreased, reaching a maximum value of −0.5 to 0.5. Taking into account the constant composition of all reaction media with regard to the polarity of solvents, these results could be explained by both the enzyme–solvent interactions and the ability of solvents to reach a critical dissolved trimethylammonium alcohol concentration. Even if the most hydrophilic solvents can easily dissolve these substrates, the increase in solvent polarity could enhance the loss of essential water molecules from the enzyme microenvironment, increasing the enzyme solvent–interaction, which may result in enzyme deactivation (Klivanov, 2001; Levitsky et al., 2000). Most hydrophobic solvents can preserve the essential water-shell around the enzyme, permitting the expression of its activity, but are not able to dissolve the nucleophile substrate and so the synthetic reaction does not occur. However, the polar solvents with a limited ability to dissolve substrates and intermediate water-mimicking properties (e.g., tertiary alcohols) exhibited the best results with regard to synthetic activity and stability. CONCLUSIONS Immobilized Candida antarctica lipase B was able to catalyze ester synthesis from vinyl ester and trimethylammonium alcohols (e.g., choline and L-carnitine) by a transesterification mechanism. The extremely high polarity of
Figure 4. Influence of hydrophobicity of the reaction media (measured by Log P) on the initial enzyme activity for butyryl choline (BuCho) (䉱) and butyryl-L-carnitine (BuCar) (䊏) synthesis catalyzed by Novozyme 435 at 40°C. See Table I for the list of solvents and Figure 2 for other reaction conditions.
these alcohols requires extremely anhydrous conditions for all the components of the reaction media and the presence of molecular sieves to limit the hydrolytic activity of the enzyme. Indeed, the synthetic activity of the enzyme was controlled by the kinetic consumption of free water molecules by the hydrolytic activity. Substrates need not be fully soluble in the bulk reaction media, and only a critical amount of soluble trimethylammonium alcohol is necessary. Neither non-water-miscible solvents (e.g., hexane) nor water mimics (e.g., formamide) were suitable for the lipasecatalyzed synthesis of trimethylammonium esters. Novozyme 435 in a midrange polar solvent (Log P of −0.5 to 0.5) exhibited the best synthetic activity, which, in the case of choline ester, was 100-fold higher than for L-carnitine ester. These results clearly show how the synthetic action of lipases could be enhanced by an adequate microenvironment. Mirta Daz was a postdoctoral fellow from the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Repu´blica Argentina.
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