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Enzymatic synthesis of ascorbyl palmitate in ultrasound-assisted system: Process optimization and kinetic evaluation
Ultrasonics Sonochemistry 18 (2011) 988–996
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Enzymatic synthesis of ascorbyl palmitate in ultrasound-assisted system: Process optimization and kinetic evaluation Lindomar A. Lerin a, Miriam C. Feiten b, Aline Richetti b, Geciane Toniazzo b, Helen Treichel b, Marcio A. Mazutti b, J. Vladimir Oliveira b, Enrique G. Oestreicher a, Débora de Oliveira b,⇑ a b
Instituto de Química – IQ/UFRJ, CT, Bloco A, Rio de Janeiro 21945-900, RJ, Brazil Universidade Regional Integrada do Alto Uruguai e das Missões, URI – Campus de Erechim, Av. Sete de Setembro, 1621, 99700-000 Erechim, RS, Brazil
a r t i c l e
i n f o
Article history: Received 26 June 2010 Received in revised form 27 September 2010 Accepted 23 December 2010 Available online 4 January 2011 Keywords: Biocatalysis Neural network Lipase Ascorbyl palmitate Experimental design Ultrasound
a b s t r a c t This work is focused on the optimization of reaction parameters for the synthesis of ascorbyl palmitate catalyzed by Candida antarctica lipase in different organic solvents under ultrasound irradiation. The sequential strategy of experimental design proved to be useful in determining the optimal conditions for reaction conversion in tert-butanol system using Novozym 435 as catalyst. The optimum production was achieved at 70 °C, ascorbic acid to palmitic acid molar ratio of 1:9, enzyme concentration of 5 wt% at 3 h of reaction, resulting in an ascorbyl palmitate conversion of about 27%. Reaction kinetics for ascorbyl palmitate production in ultrasound device showed that satisfactory reaction conversions (26%) could be achieved in short reaction times (2 h). The empirical kinetic model proposed is able to satisfactorily represent and predict the experimental data. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Enzymes are increasingly becoming the industrial workhorse for a range of processes, such as biocatalysis and bioremediation, with the aim of reducing energy and raw material consumption and amounts of waste and toxic side products [1]. An ultrasound wave, which is a periodic pressure fluctuation, can control the enzyme characteristics by altering its structure in response to a dynamic perturbation [2]. Ultrasound irradiation, a mechanical rather than an electromagnetic wave, is an alternative method to reduce mass transfer limitations in enzymatic reactions [3–5]. Ultrasonic action in liquids can cause effects of cavitation. When cavitation bubbles collapse near the phase boundary of two immiscible liquids, the resultant shock wave can provide a very efficient stirring/mixing of the layers. Because the effects of cavitation can enhance heterogeneous reactions and readily form transient reactive species, ultrasound is also a useful tool in enzymatic reactions [2,5]. Ultrasound is also known to perturb weak interactions and to induce conformational changes in protein structures [6,7]. For reactions under ultrasound irradiation, ultrasonic power is an important influencing factor. Ultrasonic waves of low intensity have a small effect on the mass transfer of the solution in compar⇑ Corresponding author. Fax: +55 54 35209090. E-mail address: [email protected] (D. de Oliveira). 1350-4177/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2010.12.013
ison to high intensity, which increases mass transfer. On the other hand, high intensity-ultrasound can lead to disruption of the enzyme [8]. These results are somewhat in compliance with some reports in the sense that increasing ultrasonic power in an appropriate range enhances enzymatic reaction rate [9,10] which was considered probably due to a decrease in substrate inhibition and aggregation based on hydrogen bonding of molecules. However, too high ultrasonic intensities were reported to reduce the activity or even inactivate the enzyme [11,12]. Ultrasound is very effective at dispersing material present in solution [13]. The application of ultrasound, therefore, will contribute to a more homogeneous reaction mixture and facilitate dispersion of lipase through substrate media, reducing agglomeration so that the reaction rate does not decrease with the increase of lipase concentration [14]. The synthesis of esters of ascorbic acid by lipase-catalyzed reactions has become a matter of great current commercial interest due to the steady growing demand for natural materials. An optimized enzymatic synthesis of L-ascorbyl esters with improved yield at reduced cost would be more appealing to the consumer and would bring benefits to the manufacturers [15]. In this sense, due to the low miscibility of the substrates, the application of ultrasound irradiation to this reaction system may lead to high reaction rates. The importance of enzymatic synthesis catalyzed by lipases to produce L-ascorbyl esters via esterification in water-miscible
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organic solvents has been emphasized in several works [16,17]. Palmitic, caprilic, capric, lauric, and stearic acids have been widely used as acyl donors. Among the unsaturated acids, one could mention oleic, linolenic, arachidonic, and docosahexaenoic acids [18]. Based on the above mentioned information, the present work is focused on the optimization of reaction conditions for ascorbyl palmitate synthesis under ultrasound irradiation by a sequential strategy of experimental planning. The kinetics for this reaction system was also investigated at the maximized experimental conditions obtained from the execution of the experimental design. To the best of our knowledge, a systematic study for maximizing the product synthesis as well as experimental data and kinetic modeling concerning the enzymatic esterification of ascorbic acid in ultrasound-assisted system has not been reported.
2. Materials and methods 2.1. Materials The substrates used in the esterification reactions were commercial palmitic acid (Vetec, 98% purity) and L-(+)-ascorbic acid (Vetec, 99% purity). Novozym 435, a lipase from Candida antarctica, purchased from Novozymes S/A (Araucária-PR-Brazil), was used as a catalyst. This enzyme was immobilized on a macroporous anionic resin, and presents a water content of 1.4 wt% (Karl Fischer titration method, DL 50, Mettler-Toledo) and an initial enzyme activity of around 60 U/g of support, determined as the initial rates in esterification reactions between lauric acid and propanol at a molar ratio of 3:1, 60° C and enzyme content of 5 wt% in relation to the substrates [19]. Acetone, tert-butanol, acetic acid, ethanol, acetone, n-propanol, and methanol of HPLC grade were tested as organic solvents (Vetec). The standard of 6-J-palmitoyl-L-ascorbic acid was obtained from Sigma–Aldrich (Fluka, 99% purity). 2.2. Equipment Experiments were carried out in a reactor with a thermostatic water bath (temperature accuracy of ±0.5° C). The experimental setup consists of an ultrasonic bath (Unique Inc.-model USC 1800A, Brazil, BR) equipped with a transducer having longitudinal vibrations. The ultrasonic unit has an operating frequency of 37 kHz and a maximum rated electrical power output of 132 W. The ultrasonic transducer (surface area of 282.2 cm2) is fitted at the bottom of the bath horizontally along the length of bath. The advantage of using such a system is that if offers much larger effective cavitational area compared to the conventional immersionbased axial transducers and hence results in uniform cavitational activity distribution in the ultrasonic bath. 2.3. 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 (0.04 N). After the addition of the enzyme to the substrates, the mixture was kept at 60° C for 15 min. The lauric acid consumption was then determined. One unit of activity (U) was defined as the amount of enzyme necessary to consume 1 lmol of lauric acid per minute. All enzymatic activity determinations were replicated at least three times.
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2.4. Products quantification Quantitative analyses of the products were conducted using an HPLC system from Agilent Series, equipped with a refractive index detector. The following instrumentation and conditions were used: Zorbax C18 column (4.6 m 250 mm, 5 lm), flow rate of 1.0 mL/ min, column temperature of 35° C; the mobile phase, acetone:methanol:H2O with 0.5% of acid acetic (75:25:5, v/v/v). The mobile phase was used as a sample dissolving solvent, and the injection volume was 20 lL. Quantification was carried out using authentic standards of ascorbyl palmitate (6-O-palmitoyl-L-ascorbic acid). Calibration curves were built with the product concentrations of 240, 480, 960, 1920, 2880, 3840, and 4800 ppm. Reaction conversion was calculated based on the content of ascorbyl palmitate in the analyzed sample and the reaction stoichiometry. 2.5. Preliminary tests At first, for all experimental runs, the enzyme was activated in an oven at 40° C for 60 min. Batch reactions were carried out in 125 cm3 conical flasks. In this initial step, some specific conditions were tested as following: use of solvent (10 mL of tert-butanol) without agitation; use of solvent with mechanical stirring (250 rpm); solvent-free system without agitation and solvent-free system with mechanical stirring (250 rpm). All experiments were then carried out under sonochemical irradiation of 132 W, temperature of 70° C, substrates molar ratio of 1:9, enzyme concentration of 5 wt% [20]. For the evaluated system that presented higher ascorbyl palmitate yields, a kinetic experiment, at the same experimental conditions cited before, was carried out to determine the reaction time to be fixed in the experimental design step. 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 reaction medium and washed twice with 10 mL of n-hexane. The recuperated enzyme was kept in a desiccator for 24 h and, after this period, the enzyme activity was determined, following the methodology proposed above. 2.6. Sequential strategy of experimental designs With the objective of determining the best reaction conditions, in the first experimental design the effects of substrates molar ratio (1:1–1:6), enzyme concentration (5–15 wt%), solvent volume (5– 15 mL), power ultrasound amplitude (52–132 W) and temperature (40–70° C), were evaluated in terms of ascorbyl palmitate production. For this experimental planning, the levels of variables were defined from preliminary experiments and the reaction time was constant at 3 h. Three replicates at the central point of each experimental planning were carried out in order to determine the experimental error. After analyzing the results of the first experimental design, a central composite rotatable design 22 (CCRD) was carried out adjusting the substrates molar ratio (1:5–1:13) and temperature (50–80° C) [21,22]. 2.7. Kinetic study of enzymatic esterification Taking into account the results obtained in the experimental designs, reaction kinetic experiments were performed adopting ascorbic acid to palmitic acid molar ratios of 1:1, 1:3, 1:6, and 1:9, enzyme concentration of 1, 5, 10, and 20 wt% (based on the total amount of substrates – ascorbic acid and palmitic acid), temperature of 50, 60, 70, and 80° C. Samples were taken from the bulk reactive system at 30 min, and 1, 2, 3, 4, and 5 h.
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0.98, respectively. The hybrid model was developed and implemented in FORTRAN 90 language.
2.8. Hybrid model formulation This study was concerned on the development of a hybrid model to predict the ascorbyl palmitate (AP) production. The model consists of a mass balance for the ascorbyl palmitate production in the batch reactor with no density and volume changes according to the following equation:
d½AP ¼v dt
ð1Þ
The main difficult in the above model is to determine the reaction rate (v), since there no data concerning the kinetics of the substrates employed. As an alternative, a feed-forward neural network (ANN) is used to predict the reaction rate in Eq. (1). The ANN was employed with the following topology: the inputs for ANN were reaction time, temperature, initial enzyme concentration, palmitic acid and ascorbic acid concentrations; only one hidden layer was used and the number of nodes in this layer was determined by trial and error; the transfer function employed was the hyperbolic tangent in the output and hidden layer; the output layer was composed of one node related to the reaction rate of ascorbyl palmitate production. The above inputs were combined to minimize the objective function F defined according to Eq. (2).
F¼
j¼NPE X
ð½APexp ½APcalc Þ2 j j
2.9. Statistical analysis The statistical analysis related to the estimated effects of each variable and process optimization was performed using the global error and the relative standard deviation between experimental data and calculated values. It may be important to mention that the kinetic results subsequently presented in this work are in fact mean values of triplicate runs, which resulted in an overall absolute deviation of reaction conversion of around 2%. The quality of the hybrid model formulation was verified using the correlation coefficient (r). All analysis was performed using the software Statistica version 6.0 (Statsoft Inc., USA). 3. Results and discussion 3.1. Preliminary tests Table 1 presents the results obtained in the preliminary tests. One can observe that the highest conversion (25.07%) was obtained using organic solvent without agitation. A comparable conversion (20.86%) was obtained from run two, after 2 h of reaction, using tert-butanol as solvent and 250 rpm. In the reaction systems without solvent, no conversion of substrates was observed. Taking into
ð2Þ
J¼1
where NPE is the number of experimental data points where the F is calculated, and ½APexp and ½APcalc represent the experimental and j j calculated ascorbyl palmitate concentrations, respectively. During the training procedure, the weights and the bias were optimized using the Simulated Annealing algorithm combined with the Nelder and Mead [23] algorithm. The parameters adopted for the Simulated Annealing algorithm as the initial artificial annealing temperature (TA) and cooling rate (a) were 10.0 and
Table 1 Results of ascorbyl palmitate yield (%) for the preliminary tests in ultrasound device (37 kHz and 132 W) using 5 wt% of Novozym 435, ascorbic acid to palmitic acid molar ratio of 1:9, 70 °C and 2 h of reaction. Reaction condition
Ascorbyl palmitate yield (%)
Tert-butanol without agitation Tert-butanol and 250 rpm Without solvent Without solvent and 250 rpm
25.07 20.86 0 0
Fig. 1. Kinetic experiment in ultrasound assisted system in organic medium in terms of ascorbyl palmitate conversion and residual enzyme activity. Experimental condition of ascorbic acid to palmitic acid molar ratio of 1:9, 10 mL of solvent, 5 wt% of Novozym 435 and 70° C.
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L.A. Lerin et al. / Ultrasonics Sonochemistry 18 (2011) 988–996 Table 2 Matrix of the Plackett–Burman experimental design (coded and real values) for ascorbyl palmitate production under sonochemical irradiation after 2 h of reaction.
a
Trial
Temperature (°C)
Enzyme concentration (wt%)
Solvent volume (mL)
Substrates molar ratioa
Ultrasonic power (W)
Yield (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
70 (1) 70 (1) 40 (1) 70 (1) 70 (1) 70 (1) 40 (1) 40 (1) 40 (1) 70 (1) 40 (1) 40 (1) 55 (0) 55 (0) 50 (0)
5 (1) 15 (1) 15 (1) 5 (1) 15 (1) 15 (1) 15 (1) 5 (1) 5 (1) 5 (1) 15 (1) 5 (1) 10 (0) 10 (0) 10 (0)
15 (1) 5 (1) 15 (1) 15 (1) 5 (1) 15 (1) 15 (1) 15 (1) 5 (1) 5 (1) 5 (1) 5 (1) 10 (0) 10 (0) 10 (0)
1:1 (1) 1:6 (1) 1:1 (1) 1:6 (1) 1:6 (1) 1:1 (1) 1:6 (1) 1:6 (1) 1:6 (1) 1:1 (1) 1:1 (1) 1:1 (1) 1:3 (0) 1:3 (0) 1:3 (0)
52 (1) 52 (1) 132 (1) 52 (1) 132 (1) 132 (1) 52 (1) 132 (1) 132 (1) 132 (1) 52 (1) 52 (1) 92 (0) 92 (0) 92 (0)
7.28 22.68 7.66 24.66 22.99 12.07 7.63 16.42 13.58 10.23 2.40 0.37 13.68 13.94 16.64
Ascorbic acid:palmitic acid.
account the results obtained here, further studies were carried out in tert-butanol system without agitation. Previous literature relates enzymatic synthesis using ultrasound devices in organic and solvent-free systems [14,24]. In general, these works show the influence of ultrasound on reaction conversion. For example, Babicz et al. [24] verified that mechanical agitation is an important factor for the production of mono- and diglycerides by enzymatic hydrolysis of soybean oil, but that this variable can also damage the enzyme support or provoke the adherence of the enzyme in the reactor. The authors obtained higher conversions at 700 rpm using Lipozyme TL IM and Lipozyme 87 IM as catalysts. Xiao et al. [25] obtained conversions of 98% after 2 h and 120 W of ultrasound, and only 48% of conversion was observed without agitation under the same experimental conditions. The authors also observed that the ultrasound did not alter the regio-selectivity of the catalyst. Fig. 1 presents the kinetic experiment in ultrasound-assisted system in organic medium in terms of ascorbyl palmitate yield and residual enzyme activity. This experiment was carried out to
define the reaction time to be fixed in the experimental design step. The experimental condition of ascorbic acid to palmitic acid molar ratio of 1:9, 10 mL of solvent, 5 wt% of Novozym 435 and 70° C was tested here. From this figure one can see that the conversion improves as a function of reaction time, reaching a maximum (25.11%) after 4 h. Related to the residual esterification activity, it can be observed that no significant activity losses were verified, with a maximum loss of 20.75%. Further studies for optimization of process conversion were performed at 3 h of reaction. Liu et al. [14] observed in the hydrolysis of soybean oil in a solvent-free system in an ultrasound device that a reaction yield of 94% at 5 h was achieved. The kinetic evaluation of hydrolysis of soybean oil catalyzed by Lipozyme TL IM in ultrasound system afforded a diglyceride yield of 40% after 1.5 h [24]. 3.2. Experimental design After determining the preliminary experimental condition that afforded the highest conversion, a Plackett–Burman design for ascorbyl palmitate production was carried out (Table 2) keeping
Fig. 2. Pareto chart of the effects of ascorbic acid to palmitic acid molar ratio and temperature on the ascorbyl palmitate production (p < 0.05). Experimental data and conditions shown in Table 2.
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Table 3 Matrix of the second experimental design (coded and real values) with responses in terms of ascorbyl palmitate conversion.
a b
Trial
Temperature (°C)
Molar ratioa
Experimental conversion (%)
Predicted conversion (%)
Y Y RED (%)b RED expY expmodel 100
1 2 3 4 5 6 7 8 9 10 11
1 (54.4) 1 (75.6) 1 (54.4) 1 (75.6) 1.41 (50) 1.41 (80) 0 (65) 0 (65) 0 (65) 0 (65) 0 (65)
1 (1:6) 1 (1:6) 1 (1:12) 1 (1:12) 0 (1:9) 0 (1:9) 1.41 (1:5) 1.41 (1:13) 0 (1:6) 0 (1:6) 0 (1:6)
11.08 19.85 9.08 21.35 13.25 27.16 15.01 16.93 23.45 21.34 21.74
11.81 21.81 11.39 21.75 11.66 26.01 14.73 14.47 22.18 22.18 22.18
6.58 9.87 25.44 1.87 11.97 4.20 1.85 14.48 5.41 3.93 2.02
Ascorbic acid:palmitic acid. RED = relative error deviation.
the reaction time fixed at 3 h, evaluating the effect of temperature, enzyme concentration, volume of solvent, substrates molar ratio and ultrasound power by means the Pareto chart on product yield. From this table one can observe that the highest conversions were obtained at 70° C and substrates molar ratio of 1:6. Experimental data appearing in Table 2 were statistically treated and Fig. 2 presents the standard Pareto chart [22], which shows the effects of reaction variables (substrates molar ratio, temperature, ultrasonic power, enzyme concentration, and solvent volume) on ascorbyl palmitate conversion. An inspection of this figure shows that the substrates molar ratio and temperature present a positive significant effect (p < 0.05) on the ascorbyl palmitate yield, making it possible to conclude that an increase in this variable leads to higher conversions. The volume of solvent, enzyme concentration and ultrasound power did not affect the ascorbyl palmitate conversion. Taking into account the results obtained in the first experimental design, a central composite rotatable design (CCRD) 22 was carried out, keeping constant the enzyme concentration (5 wt%), volume of solvent (5 mL), ultrasound power (132 W) and reaction time (3 h), varying the temperature and substrates molar ratio. Table 3 presents the matrix of the experimental design with the responses in terms of ascorbyl palmitate production. The highest conversion (27.16%) was obtained in experiment four, corresponding to a temperature of 80° C and substrates molar ratio of 1:9, showing the positive effect of these variables on process conversion. Results obtained in the second experimental design were also statistically treated and permitted the validation of an empirical coded model for ascorbyl palmitate conversion in terms of substrates molar ratio and temperature. The empirical model was validated by variance analysis (ANOVA). The R-value and F-test for regression showed that the model (Eq. (1)) was able to well represent the experimental data of ascorbyl palmitate conversion in the range of variables evaluated, allowing the development of the response surface presented in Fig. 3. This provides a satisfactory representation of the process by the empirical model, as illustrated by the predicted conversion (column five of Table 3) and the standard deviation (RED) (column six of Table 3) [22]:
Ascorbyl palmitate ¼ 22:18 þ 5:09 T 1:68 T 2 0:09 RM 3:81 RM2 þ 0:12 T RM
ð3Þ
where T denotes system temperature (°C) and MR the substrates molar ratio. An analysis of Fig. 3 permits us to verify the positive effect of temperature and substrates molar ratio on reaction conversion (maximum of 27.16%). However, increase in the substrate molar ratio (>1:9) led to a reduction in the process conversion (Table 3).
Fig. 3. Response surface (a) and contour curve (b) for ascorbyl palmitate conversion in function of temperature and ascorbic acid to palmitic acid molar ratio in ultrasound system.
This may be explained in that an excess of palmitic acid in organic phase may induce conformational changes in the enzyme structure, limiting the access of ascorbic acid to the active site of the enzyme [20]. Lerin et al. [20] evaluated the same experimental system presented here, without the use of ultrasonic irradiation. The optimum production was achieved at an ascorbic acid to palmitic acid mole ratio of 1:9, a stirring rate of 150 rpm, a temperature of 70° C, an enzyme concentration of 5 wt%, and 17 h of reaction, resulting in an ascorbyl palmitate conversion of about 67%. Although no work was found in the literature regarding the production of ascorbyl palmitate under ultrasonic irradiation by a systematic methodology of experimental design, several studies exist concerning the use of ultrasound in the improvement of esterification and hydrolysis reactions catalyzed by lipases [9,10,24–27]. 3.3. Kinetic study of enzymatic production of ascorbyl palmitate The effects of ascorbic acid to palmitic acid molar ratio, temperature and enzyme concentration were investigated on the kinetics of ascorbyl palmitate production in the ultrasound-assisted system.
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Fig. 4. Kinetics of ascorbyl palmitate production at (a) ascorbic acid to palmitic acid molar ratio of 1:1 and 1:3, (b) ascorbic acid to palmitic acid molar ratio of 1:6 and 1:9. The reaction was carried out at 70° C, 5 mL of solvent and ultrasound power of 132 W, except for when the kinetic study of the referred variable was performed.
3.4. Effect of acid to alcohol molar ratio To evaluate the effect of ascorbic acid to palmitic acid molar ratio on ascorbyl palmitate yield, the temperature was fixed at 70° C, the enzyme concentration was set at 5 wt%, 5 mL of solvent were used, and the ultrasound power was set at 132 W, making it possible to build the experimental curves of reaction conversion versus reaction time, as presented in Fig. 4a and b. These figures also present the empirical kinetic model fitting obtained using the proposed hybrid neural network approach. From this figure one can observe that similar conversions were obtained at higher molar ratios. The molar ratio of 1:1 produced lower conversions. These results are in agreement with those obtained in the experimental design that pointed out the molar ratio of 1:9 as being the most appropriate for this system after 3 h of reaction.
The substrate molar ratio is usually one of the most important parameters in enzymatic esterification reactions. Since the reaction is reversible, an enhancement in the concentration of one substrate (particularly, the palmitic acid) can displace the chemical equilibrium to products formation, resulting in higher conversions. On the other hand, high palmitic acid concentrations may reduce the reaction rate due to the inhibition effect [15,28–30]. No data could be found in the literature regarding the effect of substrate molar ratio on enzymatic esterification for ascorbyl palmitate production under an ultrasound-assisted system. The kinetic behavior obtained here can be directly compared to that of Lerin et al. [20] for the same experimental system under mechanical agitation. Similar results were observed by Burham et al. [28], where higher conversions (about 70%) in palm-based ascorbyl esters were obtained at a molar ratio of 1:8. However, for a molar ratio value of palm oil higher than eight, reduction in production of
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Fig. 5. Kinetics of ascorbyl palmitate production at (a) enzyme concentration of 1 and 5 wt%, (b) enzyme concentration of 10 and 20 wt%. The reaction was carried out at 70° C, ascorbic acid to palmitic acid mole ratio of 1:9, 5 mL of solvent and ultrasound power of 132 W.
palm based ascorbyl esters was observed. This system was also investigated under mechanical agitation. Another result using the same procedure of agitation is due to Chang et al. [15], who studied the enzymatic esterification of ascorbyl laurate and observed that an enhancement on enzyme concentration and substrate molar ratio produced a conversion of about 80% (180 rpm, 45° C, 3 mL of acetonitrile, 6 h). Lv et al. [29] obtained the maximum conversion in ascorbyl benzoate (79.48%) at a molar ratio of 10:1, and higher molar ratios were not tested due to the solubility limitations. These results indicate that an excess of L-ascorbic acid may have a stronger promotion effect on esterification than an excess of benzoic acid. Hsieh et al. [30] also evaluated the effect of the substrates molar ratio for the synthesis of ascorbyl palmitate by surfactantcoated lipase and observed higher conversions (47%) at mole ratios of 1:6, 24 h and 50° C.
3.5. Effect of enzyme concentration The effect of enzyme concentration on ascorbyl palmitate yield in an ultrasound-assisted system was evaluated at 70° C keeping the ascorbic to palmitic acid molar ratio constant at 1:9, 5 mL of solvent and ultrasound power of 132 W, varying the enzyme concentration of 1, 5, 10, and 20 wt% (based on the substrates amount). Fig. 5a and b shows the experimental data and kinetic modeling results obtained in this step. When using these enzyme concentrations it can be observed that similar initial reaction rates were obtained, leading to high conversions in short reaction times. The use of 5, 10, and 20 wt% of Novozym 435 does not present a significant difference with regard to ascorbyl palmitate production. On the other hand, at the experimental condition with 1wt% of enzyme, lower conversions were achieved, reaching 16.10%
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Fig. 6. Kinetics of ascorbyl palmitate production at temperatures of 50, 60, 70, and 80° C. The reaction was carried out at ascorbic acid to palmitic acid molar ratio of 1:9, 5 mL of solvent and ultrasound power of 132 W.
conversion after 3 h. From Fig. 5 one can also observe that the highest conversion was obtained using 20 wt% of enzyme and 4 h of reaction, resulting in a conversion of about 33%. Fig. 5b shows that the use of 10 and 20 wt% of enzyme do not affect significantly the reaction conversion. A possible explanation might be related to the fact that an excess of enzyme in the reaction medium could not contribute to the conversion enhancement, since high enzyme concentration may form aggregates, thus not making the enzyme active site available to the substrate. The enzyme molecules on the 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 [31]. Babicz et al. [24] evaluated the effect of enzyme concentration (1 and 2 wt%) on the hydrolysis of soybean oil in ultrasound system (47 kHz and 125 W) and obtained conversions in diglycerides of 40% using 1 wt% of Lipozyme TL IM after 1.5 h of reaction. The use of 2 wt% led to conversions of 32% in 3 h. Liu et al. [14] studied the effect of enzyme concentration on the hydrolysis of soybean oil at 0.1, 0.55, 1, 2, and 3 wt% of lipase from C. lipolytica in a solvent-free system using ultrasound-assisted system. Reaction rates showed a slight enhancement as a function of enzyme concentration. The authors observe a reduction in reaction rate with an increase in enzyme concentration. Based on the results obtained, the authors believe that there is a critical enzyme concentration (0.55 wt%, in this case), above that the reaction interface becomes saturated.
From this figure one can observe that at 80° C, after 3 h of reaction, an ascorbyl palmitate yield of 35.06% was obtained. At 50, 60, and 70° C similar behavior was verified. This result seems to be coherent with those obtained in the experimental design step. Liu et al. [14] evaluated the influence of temperature on hydrolysis of soybean oil catalyzed by a lipase from C. lipolytica in solvent-free system using ultrasonic energy. The authors reported that good hydrolytic activities were reached from 30 to 55° C; at 65° C the enzyme lost all its activity. 4. Conclusions New experimental data on ultrasound-assisted enzymatic esterification for ascorbyl palmitate production is reported in this work, showing a perspective of the technique to overcome mass transfer limitations arising from the use of ascorbic acid as substrate. The use of tert-butanol without mechanical agitation in an ultrasoundassisted system led to satisfactory conversions in ascorbyl palmitate. The optimum production (27.15%) was achieved at ascorbic acid to palmitic acid molar ratio of 1:9, ultrasound power of 132 W, 80° C, Novozym 435 concentration of 5 wt% at 3 h of reaction. The reaction kinetics for ascorbyl palmitate production showed that good reaction conversions (26%) could be achieved in a short reaction time (2 h). The empirical kinetic model proposed proved to be capable of satisfactorily representing the experimental data. Acknowledgements The authors thank CNPq and CAPES for the financial support and scholarships.
3.6. Effect of temperature References To evaluate the effect of temperature (50, 60, 70, and 80° C) on ascorbyl palmitate yield under ultrasonic energy, the molar ratio of ascorbic to palmitic acid was kept fixed at 1:9, enzyme concentration at 5 wt%, 5 mL of solvent and ultrasound power of 132 W, making it possible to follow the course of the reaction conversion, as presented in Fig. 6. This figure also shows good agreement with the results obtained using the proposed empirical kinetic model.
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[18] S.K. Karmee, Biocatalytic synthesis of ascorbyl esters and their biotechnological applications, Appl. Microbiol. Biotechnol. 81 (2009) 1013– 1022. [19] D. Oliveira, A.C. Feihrmann, C. Dariva, A.G. Cunha, J.V. Bevilaqua, J. Destain, J.V. Oliveira, D.M.G. Freire, Influence of compressed fluids treatment on the activity of Yarrowia lipolytica lipase, J. Mol. Catal. B Enzym. 39 (2006) 117–123. [20] L.A. Lerin, A. Richetti, R.M. Dallago, H. Treichel, M.A. Mazutti, J.V. Oliveira, O.A.C. Antunes, E.G. Oestreicher, D. de Oliveira, Enzymatic synthesis of ascorbyl palmitate in organic solvents: process optimization and kinetic evaluation, Food Biop. Technol., accepted for publication. doi:10.1007/s11947010-0398-1. [21] M.I. Rodrigues, A.F. Iemma, Planejamento de experimentos e otimização de processos, Editora Casa do Pão, Campinas, 2006. [22] D.C. Montgomery, Design and Analysis of Experiments, John Wiley and Sons, New York, 1991. [23] W.H. Press, S.A. Teukolsky, W.T. Vetterling, B.P. Flannery, Numerical Recipes in FORTRAN, Cambridge University Press, USA, 1992. [24] I. Babicz, S.G.F. Leite, R.O.M.A. De Souza, O.A.C. Antunes, Lipase-catalyzed diacylglycerol production under sonochemical irradiation, Ultrason. Sonochem. 17 (2010) 4–6. [25] Y.-M. Xiao, Q. Wu, Y. Caia, X. Lin, Ultrasound-accelerated enzymatic synthesis of sugar esters in nonaqueous solvents, Carbon Res. 340 (2005) 2097–2103. [26] Z.-G. Chen, M.-H. Zong, Z.-X. Gu, Y.-B. Han, Effect of ultrasound on enzymatic acylation of konjac glucomannan, Biophys. Biosyst. Eng. 31 (2008) 351–356. [27] K. Zhu, H. Liu, P. Han, P. Wei, Study of ultrasound-promoted lipase-catalyzed synthesis of fructose ester, Front Chem. Eng. China. (2010). doi:10.1007/ s11705-009-0312-4. [28] H. Burham, R.A.G.A. Rasheed, N.M. Noor, S. Badruddin, H. Sidek, Enzymatic synthesis of palm-based ascorbyl esters, J. Mol. Catal. B Enzym. 58 (2009) 153– 157. [29] L.-X. Lv, Y. Pan, Y.-Q. Li, Synthesis of ascorbyl benzoate in organic solvents and study of its antioxygenic and antimicrobial properties, Food Chem. 101 (2007) 1626–1632. [30] H. Hsieh, G.R. Nair, W. Wu, Production of ascorbyl palmitate by surfactantcoated lipase in organic media, J. Agric. Food Chem. 54 (2006) 5777–5781. [31] M. Karra-Châabouni, H. Ghamghi, S. Bezzine, A. Rekik, Y. Gargouri, Production of flavour esters by immobilized Sthaphylococcus simulans lipase in a solventfree system, Proc. Biochem. 41 (2006) 1692–1698.
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Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultsonch
Enzymatic synthesis of ascorbyl palmitate in ultrasound-assisted system: Process optimization and kinetic evaluation Lindomar A. Lerin a, Miriam C. Feiten b, Aline Richetti b, Geciane Toniazzo b, Helen Treichel b, Marcio A. Mazutti b, J. Vladimir Oliveira b, Enrique G. Oestreicher a, Débora de Oliveira b,⇑ a b
Instituto de Química – IQ/UFRJ, CT, Bloco A, Rio de Janeiro 21945-900, RJ, Brazil Universidade Regional Integrada do Alto Uruguai e das Missões, URI – Campus de Erechim, Av. Sete de Setembro, 1621, 99700-000 Erechim, RS, Brazil
a r t i c l e
i n f o
Article history: Received 26 June 2010 Received in revised form 27 September 2010 Accepted 23 December 2010 Available online 4 January 2011 Keywords: Biocatalysis Neural network Lipase Ascorbyl palmitate Experimental design Ultrasound
a b s t r a c t This work is focused on the optimization of reaction parameters for the synthesis of ascorbyl palmitate catalyzed by Candida antarctica lipase in different organic solvents under ultrasound irradiation. The sequential strategy of experimental design proved to be useful in determining the optimal conditions for reaction conversion in tert-butanol system using Novozym 435 as catalyst. The optimum production was achieved at 70 °C, ascorbic acid to palmitic acid molar ratio of 1:9, enzyme concentration of 5 wt% at 3 h of reaction, resulting in an ascorbyl palmitate conversion of about 27%. Reaction kinetics for ascorbyl palmitate production in ultrasound device showed that satisfactory reaction conversions (26%) could be achieved in short reaction times (2 h). The empirical kinetic model proposed is able to satisfactorily represent and predict the experimental data. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Enzymes are increasingly becoming the industrial workhorse for a range of processes, such as biocatalysis and bioremediation, with the aim of reducing energy and raw material consumption and amounts of waste and toxic side products [1]. An ultrasound wave, which is a periodic pressure fluctuation, can control the enzyme characteristics by altering its structure in response to a dynamic perturbation [2]. Ultrasound irradiation, a mechanical rather than an electromagnetic wave, is an alternative method to reduce mass transfer limitations in enzymatic reactions [3–5]. Ultrasonic action in liquids can cause effects of cavitation. When cavitation bubbles collapse near the phase boundary of two immiscible liquids, the resultant shock wave can provide a very efficient stirring/mixing of the layers. Because the effects of cavitation can enhance heterogeneous reactions and readily form transient reactive species, ultrasound is also a useful tool in enzymatic reactions [2,5]. Ultrasound is also known to perturb weak interactions and to induce conformational changes in protein structures [6,7]. For reactions under ultrasound irradiation, ultrasonic power is an important influencing factor. Ultrasonic waves of low intensity have a small effect on the mass transfer of the solution in compar⇑ Corresponding author. Fax: +55 54 35209090. E-mail address: [email protected] (D. de Oliveira). 1350-4177/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2010.12.013
ison to high intensity, which increases mass transfer. On the other hand, high intensity-ultrasound can lead to disruption of the enzyme [8]. These results are somewhat in compliance with some reports in the sense that increasing ultrasonic power in an appropriate range enhances enzymatic reaction rate [9,10] which was considered probably due to a decrease in substrate inhibition and aggregation based on hydrogen bonding of molecules. However, too high ultrasonic intensities were reported to reduce the activity or even inactivate the enzyme [11,12]. Ultrasound is very effective at dispersing material present in solution [13]. The application of ultrasound, therefore, will contribute to a more homogeneous reaction mixture and facilitate dispersion of lipase through substrate media, reducing agglomeration so that the reaction rate does not decrease with the increase of lipase concentration [14]. The synthesis of esters of ascorbic acid by lipase-catalyzed reactions has become a matter of great current commercial interest due to the steady growing demand for natural materials. An optimized enzymatic synthesis of L-ascorbyl esters with improved yield at reduced cost would be more appealing to the consumer and would bring benefits to the manufacturers [15]. In this sense, due to the low miscibility of the substrates, the application of ultrasound irradiation to this reaction system may lead to high reaction rates. The importance of enzymatic synthesis catalyzed by lipases to produce L-ascorbyl esters via esterification in water-miscible
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organic solvents has been emphasized in several works [16,17]. Palmitic, caprilic, capric, lauric, and stearic acids have been widely used as acyl donors. Among the unsaturated acids, one could mention oleic, linolenic, arachidonic, and docosahexaenoic acids [18]. Based on the above mentioned information, the present work is focused on the optimization of reaction conditions for ascorbyl palmitate synthesis under ultrasound irradiation by a sequential strategy of experimental planning. The kinetics for this reaction system was also investigated at the maximized experimental conditions obtained from the execution of the experimental design. To the best of our knowledge, a systematic study for maximizing the product synthesis as well as experimental data and kinetic modeling concerning the enzymatic esterification of ascorbic acid in ultrasound-assisted system has not been reported.
2. Materials and methods 2.1. Materials The substrates used in the esterification reactions were commercial palmitic acid (Vetec, 98% purity) and L-(+)-ascorbic acid (Vetec, 99% purity). Novozym 435, a lipase from Candida antarctica, purchased from Novozymes S/A (Araucária-PR-Brazil), was used as a catalyst. This enzyme was immobilized on a macroporous anionic resin, and presents a water content of 1.4 wt% (Karl Fischer titration method, DL 50, Mettler-Toledo) and an initial enzyme activity of around 60 U/g of support, determined as the initial rates in esterification reactions between lauric acid and propanol at a molar ratio of 3:1, 60° C and enzyme content of 5 wt% in relation to the substrates [19]. Acetone, tert-butanol, acetic acid, ethanol, acetone, n-propanol, and methanol of HPLC grade were tested as organic solvents (Vetec). The standard of 6-J-palmitoyl-L-ascorbic acid was obtained from Sigma–Aldrich (Fluka, 99% purity). 2.2. Equipment Experiments were carried out in a reactor with a thermostatic water bath (temperature accuracy of ±0.5° C). The experimental setup consists of an ultrasonic bath (Unique Inc.-model USC 1800A, Brazil, BR) equipped with a transducer having longitudinal vibrations. The ultrasonic unit has an operating frequency of 37 kHz and a maximum rated electrical power output of 132 W. The ultrasonic transducer (surface area of 282.2 cm2) is fitted at the bottom of the bath horizontally along the length of bath. The advantage of using such a system is that if offers much larger effective cavitational area compared to the conventional immersionbased axial transducers and hence results in uniform cavitational activity distribution in the ultrasonic bath. 2.3. 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 (0.04 N). After the addition of the enzyme to the substrates, the mixture was kept at 60° C for 15 min. The lauric acid consumption was then determined. One unit of activity (U) was defined as the amount of enzyme necessary to consume 1 lmol of lauric acid per minute. All enzymatic activity determinations were replicated at least three times.
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2.4. Products quantification Quantitative analyses of the products were conducted using an HPLC system from Agilent Series, equipped with a refractive index detector. The following instrumentation and conditions were used: Zorbax C18 column (4.6 m 250 mm, 5 lm), flow rate of 1.0 mL/ min, column temperature of 35° C; the mobile phase, acetone:methanol:H2O with 0.5% of acid acetic (75:25:5, v/v/v). The mobile phase was used as a sample dissolving solvent, and the injection volume was 20 lL. Quantification was carried out using authentic standards of ascorbyl palmitate (6-O-palmitoyl-L-ascorbic acid). Calibration curves were built with the product concentrations of 240, 480, 960, 1920, 2880, 3840, and 4800 ppm. Reaction conversion was calculated based on the content of ascorbyl palmitate in the analyzed sample and the reaction stoichiometry. 2.5. Preliminary tests At first, for all experimental runs, the enzyme was activated in an oven at 40° C for 60 min. Batch reactions were carried out in 125 cm3 conical flasks. In this initial step, some specific conditions were tested as following: use of solvent (10 mL of tert-butanol) without agitation; use of solvent with mechanical stirring (250 rpm); solvent-free system without agitation and solvent-free system with mechanical stirring (250 rpm). All experiments were then carried out under sonochemical irradiation of 132 W, temperature of 70° C, substrates molar ratio of 1:9, enzyme concentration of 5 wt% [20]. For the evaluated system that presented higher ascorbyl palmitate yields, a kinetic experiment, at the same experimental conditions cited before, was carried out to determine the reaction time to be fixed in the experimental design step. 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 reaction medium and washed twice with 10 mL of n-hexane. The recuperated enzyme was kept in a desiccator for 24 h and, after this period, the enzyme activity was determined, following the methodology proposed above. 2.6. Sequential strategy of experimental designs With the objective of determining the best reaction conditions, in the first experimental design the effects of substrates molar ratio (1:1–1:6), enzyme concentration (5–15 wt%), solvent volume (5– 15 mL), power ultrasound amplitude (52–132 W) and temperature (40–70° C), were evaluated in terms of ascorbyl palmitate production. For this experimental planning, the levels of variables were defined from preliminary experiments and the reaction time was constant at 3 h. Three replicates at the central point of each experimental planning were carried out in order to determine the experimental error. After analyzing the results of the first experimental design, a central composite rotatable design 22 (CCRD) was carried out adjusting the substrates molar ratio (1:5–1:13) and temperature (50–80° C) [21,22]. 2.7. Kinetic study of enzymatic esterification Taking into account the results obtained in the experimental designs, reaction kinetic experiments were performed adopting ascorbic acid to palmitic acid molar ratios of 1:1, 1:3, 1:6, and 1:9, enzyme concentration of 1, 5, 10, and 20 wt% (based on the total amount of substrates – ascorbic acid and palmitic acid), temperature of 50, 60, 70, and 80° C. Samples were taken from the bulk reactive system at 30 min, and 1, 2, 3, 4, and 5 h.
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0.98, respectively. The hybrid model was developed and implemented in FORTRAN 90 language.
2.8. Hybrid model formulation This study was concerned on the development of a hybrid model to predict the ascorbyl palmitate (AP) production. The model consists of a mass balance for the ascorbyl palmitate production in the batch reactor with no density and volume changes according to the following equation:
d½AP ¼v dt
ð1Þ
The main difficult in the above model is to determine the reaction rate (v), since there no data concerning the kinetics of the substrates employed. As an alternative, a feed-forward neural network (ANN) is used to predict the reaction rate in Eq. (1). The ANN was employed with the following topology: the inputs for ANN were reaction time, temperature, initial enzyme concentration, palmitic acid and ascorbic acid concentrations; only one hidden layer was used and the number of nodes in this layer was determined by trial and error; the transfer function employed was the hyperbolic tangent in the output and hidden layer; the output layer was composed of one node related to the reaction rate of ascorbyl palmitate production. The above inputs were combined to minimize the objective function F defined according to Eq. (2).
F¼
j¼NPE X
ð½APexp ½APcalc Þ2 j j
2.9. Statistical analysis The statistical analysis related to the estimated effects of each variable and process optimization was performed using the global error and the relative standard deviation between experimental data and calculated values. It may be important to mention that the kinetic results subsequently presented in this work are in fact mean values of triplicate runs, which resulted in an overall absolute deviation of reaction conversion of around 2%. The quality of the hybrid model formulation was verified using the correlation coefficient (r). All analysis was performed using the software Statistica version 6.0 (Statsoft Inc., USA). 3. Results and discussion 3.1. Preliminary tests Table 1 presents the results obtained in the preliminary tests. One can observe that the highest conversion (25.07%) was obtained using organic solvent without agitation. A comparable conversion (20.86%) was obtained from run two, after 2 h of reaction, using tert-butanol as solvent and 250 rpm. In the reaction systems without solvent, no conversion of substrates was observed. Taking into
ð2Þ
J¼1
where NPE is the number of experimental data points where the F is calculated, and ½APexp and ½APcalc represent the experimental and j j calculated ascorbyl palmitate concentrations, respectively. During the training procedure, the weights and the bias were optimized using the Simulated Annealing algorithm combined with the Nelder and Mead [23] algorithm. The parameters adopted for the Simulated Annealing algorithm as the initial artificial annealing temperature (TA) and cooling rate (a) were 10.0 and
Table 1 Results of ascorbyl palmitate yield (%) for the preliminary tests in ultrasound device (37 kHz and 132 W) using 5 wt% of Novozym 435, ascorbic acid to palmitic acid molar ratio of 1:9, 70 °C and 2 h of reaction. Reaction condition
Ascorbyl palmitate yield (%)
Tert-butanol without agitation Tert-butanol and 250 rpm Without solvent Without solvent and 250 rpm
25.07 20.86 0 0
Fig. 1. Kinetic experiment in ultrasound assisted system in organic medium in terms of ascorbyl palmitate conversion and residual enzyme activity. Experimental condition of ascorbic acid to palmitic acid molar ratio of 1:9, 10 mL of solvent, 5 wt% of Novozym 435 and 70° C.
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L.A. Lerin et al. / Ultrasonics Sonochemistry 18 (2011) 988–996 Table 2 Matrix of the Plackett–Burman experimental design (coded and real values) for ascorbyl palmitate production under sonochemical irradiation after 2 h of reaction.
a
Trial
Temperature (°C)
Enzyme concentration (wt%)
Solvent volume (mL)
Substrates molar ratioa
Ultrasonic power (W)
Yield (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
70 (1) 70 (1) 40 (1) 70 (1) 70 (1) 70 (1) 40 (1) 40 (1) 40 (1) 70 (1) 40 (1) 40 (1) 55 (0) 55 (0) 50 (0)
5 (1) 15 (1) 15 (1) 5 (1) 15 (1) 15 (1) 15 (1) 5 (1) 5 (1) 5 (1) 15 (1) 5 (1) 10 (0) 10 (0) 10 (0)
15 (1) 5 (1) 15 (1) 15 (1) 5 (1) 15 (1) 15 (1) 15 (1) 5 (1) 5 (1) 5 (1) 5 (1) 10 (0) 10 (0) 10 (0)
1:1 (1) 1:6 (1) 1:1 (1) 1:6 (1) 1:6 (1) 1:1 (1) 1:6 (1) 1:6 (1) 1:6 (1) 1:1 (1) 1:1 (1) 1:1 (1) 1:3 (0) 1:3 (0) 1:3 (0)
52 (1) 52 (1) 132 (1) 52 (1) 132 (1) 132 (1) 52 (1) 132 (1) 132 (1) 132 (1) 52 (1) 52 (1) 92 (0) 92 (0) 92 (0)
7.28 22.68 7.66 24.66 22.99 12.07 7.63 16.42 13.58 10.23 2.40 0.37 13.68 13.94 16.64
Ascorbic acid:palmitic acid.
account the results obtained here, further studies were carried out in tert-butanol system without agitation. Previous literature relates enzymatic synthesis using ultrasound devices in organic and solvent-free systems [14,24]. In general, these works show the influence of ultrasound on reaction conversion. For example, Babicz et al. [24] verified that mechanical agitation is an important factor for the production of mono- and diglycerides by enzymatic hydrolysis of soybean oil, but that this variable can also damage the enzyme support or provoke the adherence of the enzyme in the reactor. The authors obtained higher conversions at 700 rpm using Lipozyme TL IM and Lipozyme 87 IM as catalysts. Xiao et al. [25] obtained conversions of 98% after 2 h and 120 W of ultrasound, and only 48% of conversion was observed without agitation under the same experimental conditions. The authors also observed that the ultrasound did not alter the regio-selectivity of the catalyst. Fig. 1 presents the kinetic experiment in ultrasound-assisted system in organic medium in terms of ascorbyl palmitate yield and residual enzyme activity. This experiment was carried out to
define the reaction time to be fixed in the experimental design step. The experimental condition of ascorbic acid to palmitic acid molar ratio of 1:9, 10 mL of solvent, 5 wt% of Novozym 435 and 70° C was tested here. From this figure one can see that the conversion improves as a function of reaction time, reaching a maximum (25.11%) after 4 h. Related to the residual esterification activity, it can be observed that no significant activity losses were verified, with a maximum loss of 20.75%. Further studies for optimization of process conversion were performed at 3 h of reaction. Liu et al. [14] observed in the hydrolysis of soybean oil in a solvent-free system in an ultrasound device that a reaction yield of 94% at 5 h was achieved. The kinetic evaluation of hydrolysis of soybean oil catalyzed by Lipozyme TL IM in ultrasound system afforded a diglyceride yield of 40% after 1.5 h [24]. 3.2. Experimental design After determining the preliminary experimental condition that afforded the highest conversion, a Plackett–Burman design for ascorbyl palmitate production was carried out (Table 2) keeping
Fig. 2. Pareto chart of the effects of ascorbic acid to palmitic acid molar ratio and temperature on the ascorbyl palmitate production (p < 0.05). Experimental data and conditions shown in Table 2.
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Table 3 Matrix of the second experimental design (coded and real values) with responses in terms of ascorbyl palmitate conversion.
a b
Trial
Temperature (°C)
Molar ratioa
Experimental conversion (%)
Predicted conversion (%)
Y Y RED (%)b RED expY expmodel 100
1 2 3 4 5 6 7 8 9 10 11
1 (54.4) 1 (75.6) 1 (54.4) 1 (75.6) 1.41 (50) 1.41 (80) 0 (65) 0 (65) 0 (65) 0 (65) 0 (65)
1 (1:6) 1 (1:6) 1 (1:12) 1 (1:12) 0 (1:9) 0 (1:9) 1.41 (1:5) 1.41 (1:13) 0 (1:6) 0 (1:6) 0 (1:6)
11.08 19.85 9.08 21.35 13.25 27.16 15.01 16.93 23.45 21.34 21.74
11.81 21.81 11.39 21.75 11.66 26.01 14.73 14.47 22.18 22.18 22.18
6.58 9.87 25.44 1.87 11.97 4.20 1.85 14.48 5.41 3.93 2.02
Ascorbic acid:palmitic acid. RED = relative error deviation.
the reaction time fixed at 3 h, evaluating the effect of temperature, enzyme concentration, volume of solvent, substrates molar ratio and ultrasound power by means the Pareto chart on product yield. From this table one can observe that the highest conversions were obtained at 70° C and substrates molar ratio of 1:6. Experimental data appearing in Table 2 were statistically treated and Fig. 2 presents the standard Pareto chart [22], which shows the effects of reaction variables (substrates molar ratio, temperature, ultrasonic power, enzyme concentration, and solvent volume) on ascorbyl palmitate conversion. An inspection of this figure shows that the substrates molar ratio and temperature present a positive significant effect (p < 0.05) on the ascorbyl palmitate yield, making it possible to conclude that an increase in this variable leads to higher conversions. The volume of solvent, enzyme concentration and ultrasound power did not affect the ascorbyl palmitate conversion. Taking into account the results obtained in the first experimental design, a central composite rotatable design (CCRD) 22 was carried out, keeping constant the enzyme concentration (5 wt%), volume of solvent (5 mL), ultrasound power (132 W) and reaction time (3 h), varying the temperature and substrates molar ratio. Table 3 presents the matrix of the experimental design with the responses in terms of ascorbyl palmitate production. The highest conversion (27.16%) was obtained in experiment four, corresponding to a temperature of 80° C and substrates molar ratio of 1:9, showing the positive effect of these variables on process conversion. Results obtained in the second experimental design were also statistically treated and permitted the validation of an empirical coded model for ascorbyl palmitate conversion in terms of substrates molar ratio and temperature. The empirical model was validated by variance analysis (ANOVA). The R-value and F-test for regression showed that the model (Eq. (1)) was able to well represent the experimental data of ascorbyl palmitate conversion in the range of variables evaluated, allowing the development of the response surface presented in Fig. 3. This provides a satisfactory representation of the process by the empirical model, as illustrated by the predicted conversion (column five of Table 3) and the standard deviation (RED) (column six of Table 3) [22]:
Ascorbyl palmitate ¼ 22:18 þ 5:09 T 1:68 T 2 0:09 RM 3:81 RM2 þ 0:12 T RM
ð3Þ
where T denotes system temperature (°C) and MR the substrates molar ratio. An analysis of Fig. 3 permits us to verify the positive effect of temperature and substrates molar ratio on reaction conversion (maximum of 27.16%). However, increase in the substrate molar ratio (>1:9) led to a reduction in the process conversion (Table 3).
Fig. 3. Response surface (a) and contour curve (b) for ascorbyl palmitate conversion in function of temperature and ascorbic acid to palmitic acid molar ratio in ultrasound system.
This may be explained in that an excess of palmitic acid in organic phase may induce conformational changes in the enzyme structure, limiting the access of ascorbic acid to the active site of the enzyme [20]. Lerin et al. [20] evaluated the same experimental system presented here, without the use of ultrasonic irradiation. The optimum production was achieved at an ascorbic acid to palmitic acid mole ratio of 1:9, a stirring rate of 150 rpm, a temperature of 70° C, an enzyme concentration of 5 wt%, and 17 h of reaction, resulting in an ascorbyl palmitate conversion of about 67%. Although no work was found in the literature regarding the production of ascorbyl palmitate under ultrasonic irradiation by a systematic methodology of experimental design, several studies exist concerning the use of ultrasound in the improvement of esterification and hydrolysis reactions catalyzed by lipases [9,10,24–27]. 3.3. Kinetic study of enzymatic production of ascorbyl palmitate The effects of ascorbic acid to palmitic acid molar ratio, temperature and enzyme concentration were investigated on the kinetics of ascorbyl palmitate production in the ultrasound-assisted system.
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Fig. 4. Kinetics of ascorbyl palmitate production at (a) ascorbic acid to palmitic acid molar ratio of 1:1 and 1:3, (b) ascorbic acid to palmitic acid molar ratio of 1:6 and 1:9. The reaction was carried out at 70° C, 5 mL of solvent and ultrasound power of 132 W, except for when the kinetic study of the referred variable was performed.
3.4. Effect of acid to alcohol molar ratio To evaluate the effect of ascorbic acid to palmitic acid molar ratio on ascorbyl palmitate yield, the temperature was fixed at 70° C, the enzyme concentration was set at 5 wt%, 5 mL of solvent were used, and the ultrasound power was set at 132 W, making it possible to build the experimental curves of reaction conversion versus reaction time, as presented in Fig. 4a and b. These figures also present the empirical kinetic model fitting obtained using the proposed hybrid neural network approach. From this figure one can observe that similar conversions were obtained at higher molar ratios. The molar ratio of 1:1 produced lower conversions. These results are in agreement with those obtained in the experimental design that pointed out the molar ratio of 1:9 as being the most appropriate for this system after 3 h of reaction.
The substrate molar ratio is usually one of the most important parameters in enzymatic esterification reactions. Since the reaction is reversible, an enhancement in the concentration of one substrate (particularly, the palmitic acid) can displace the chemical equilibrium to products formation, resulting in higher conversions. On the other hand, high palmitic acid concentrations may reduce the reaction rate due to the inhibition effect [15,28–30]. No data could be found in the literature regarding the effect of substrate molar ratio on enzymatic esterification for ascorbyl palmitate production under an ultrasound-assisted system. The kinetic behavior obtained here can be directly compared to that of Lerin et al. [20] for the same experimental system under mechanical agitation. Similar results were observed by Burham et al. [28], where higher conversions (about 70%) in palm-based ascorbyl esters were obtained at a molar ratio of 1:8. However, for a molar ratio value of palm oil higher than eight, reduction in production of
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Fig. 5. Kinetics of ascorbyl palmitate production at (a) enzyme concentration of 1 and 5 wt%, (b) enzyme concentration of 10 and 20 wt%. The reaction was carried out at 70° C, ascorbic acid to palmitic acid mole ratio of 1:9, 5 mL of solvent and ultrasound power of 132 W.
palm based ascorbyl esters was observed. This system was also investigated under mechanical agitation. Another result using the same procedure of agitation is due to Chang et al. [15], who studied the enzymatic esterification of ascorbyl laurate and observed that an enhancement on enzyme concentration and substrate molar ratio produced a conversion of about 80% (180 rpm, 45° C, 3 mL of acetonitrile, 6 h). Lv et al. [29] obtained the maximum conversion in ascorbyl benzoate (79.48%) at a molar ratio of 10:1, and higher molar ratios were not tested due to the solubility limitations. These results indicate that an excess of L-ascorbic acid may have a stronger promotion effect on esterification than an excess of benzoic acid. Hsieh et al. [30] also evaluated the effect of the substrates molar ratio for the synthesis of ascorbyl palmitate by surfactantcoated lipase and observed higher conversions (47%) at mole ratios of 1:6, 24 h and 50° C.
3.5. Effect of enzyme concentration The effect of enzyme concentration on ascorbyl palmitate yield in an ultrasound-assisted system was evaluated at 70° C keeping the ascorbic to palmitic acid molar ratio constant at 1:9, 5 mL of solvent and ultrasound power of 132 W, varying the enzyme concentration of 1, 5, 10, and 20 wt% (based on the substrates amount). Fig. 5a and b shows the experimental data and kinetic modeling results obtained in this step. When using these enzyme concentrations it can be observed that similar initial reaction rates were obtained, leading to high conversions in short reaction times. The use of 5, 10, and 20 wt% of Novozym 435 does not present a significant difference with regard to ascorbyl palmitate production. On the other hand, at the experimental condition with 1wt% of enzyme, lower conversions were achieved, reaching 16.10%
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Fig. 6. Kinetics of ascorbyl palmitate production at temperatures of 50, 60, 70, and 80° C. The reaction was carried out at ascorbic acid to palmitic acid molar ratio of 1:9, 5 mL of solvent and ultrasound power of 132 W.
conversion after 3 h. From Fig. 5 one can also observe that the highest conversion was obtained using 20 wt% of enzyme and 4 h of reaction, resulting in a conversion of about 33%. Fig. 5b shows that the use of 10 and 20 wt% of enzyme do not affect significantly the reaction conversion. A possible explanation might be related to the fact that an excess of enzyme in the reaction medium could not contribute to the conversion enhancement, since high enzyme concentration may form aggregates, thus not making the enzyme active site available to the substrate. The enzyme molecules on the 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 [31]. Babicz et al. [24] evaluated the effect of enzyme concentration (1 and 2 wt%) on the hydrolysis of soybean oil in ultrasound system (47 kHz and 125 W) and obtained conversions in diglycerides of 40% using 1 wt% of Lipozyme TL IM after 1.5 h of reaction. The use of 2 wt% led to conversions of 32% in 3 h. Liu et al. [14] studied the effect of enzyme concentration on the hydrolysis of soybean oil at 0.1, 0.55, 1, 2, and 3 wt% of lipase from C. lipolytica in a solvent-free system using ultrasound-assisted system. Reaction rates showed a slight enhancement as a function of enzyme concentration. The authors observe a reduction in reaction rate with an increase in enzyme concentration. Based on the results obtained, the authors believe that there is a critical enzyme concentration (0.55 wt%, in this case), above that the reaction interface becomes saturated.
From this figure one can observe that at 80° C, after 3 h of reaction, an ascorbyl palmitate yield of 35.06% was obtained. At 50, 60, and 70° C similar behavior was verified. This result seems to be coherent with those obtained in the experimental design step. Liu et al. [14] evaluated the influence of temperature on hydrolysis of soybean oil catalyzed by a lipase from C. lipolytica in solvent-free system using ultrasonic energy. The authors reported that good hydrolytic activities were reached from 30 to 55° C; at 65° C the enzyme lost all its activity. 4. Conclusions New experimental data on ultrasound-assisted enzymatic esterification for ascorbyl palmitate production is reported in this work, showing a perspective of the technique to overcome mass transfer limitations arising from the use of ascorbic acid as substrate. The use of tert-butanol without mechanical agitation in an ultrasoundassisted system led to satisfactory conversions in ascorbyl palmitate. The optimum production (27.15%) was achieved at ascorbic acid to palmitic acid molar ratio of 1:9, ultrasound power of 132 W, 80° C, Novozym 435 concentration of 5 wt% at 3 h of reaction. The reaction kinetics for ascorbyl palmitate production showed that good reaction conversions (26%) could be achieved in a short reaction time (2 h). The empirical kinetic model proposed proved to be capable of satisfactorily representing the experimental data. Acknowledgements The authors thank CNPq and CAPES for the financial support and scholarships.
3.6. Effect of temperature References To evaluate the effect of temperature (50, 60, 70, and 80° C) on ascorbyl palmitate yield under ultrasonic energy, the molar ratio of ascorbic to palmitic acid was kept fixed at 1:9, enzyme concentration at 5 wt%, 5 mL of solvent and ultrasound power of 132 W, making it possible to follow the course of the reaction conversion, as presented in Fig. 6. This figure also shows good agreement with the results obtained using the proposed empirical kinetic model.
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