Lipase catalyzed ascorbyl palmitate synthesis under microwave irradiation

Article abstract of DOI:10.1016/j.molcatb.2014.02.002

The production of ascorbyl palmitate from palmitic acid and ascorbic acid through the esterification catalyzed by Novozym 435 was performed under microwave irradiation. The effects of different reaction parameters were evaluated. The reactions were carried out in organic solvent (acetone, tert-amyl alcohol, cyclohexane, acetonitrile) and the results show that tert-amyl alcohol was the best solvent for this reaction. Parameters as temperature, molar ratio and enzyme loading were evaluated through a central composite design (CCD). In the optimized conditions, 15% (w/w in relation of ascorbic acid weight) of enzyme, 70 C and molar ratio 1:5, 71% yield of ascorbyl palmitate could be achieved after 1 h.

Full text of DOI:10.1016/j.molcatb.2014.02.002

Accepted Manuscript
Title: Lipase Catalyzed Ascorbyl Palmitate Synthesis under
Microwave Irradiation
Author: Ingrid C.R. Costa Felipe K. Sutili Gabriela V.V. da
Silva Selma G.F. Leite Leandro S.M. Miranda Rodrigo
O.M.A. de Souza
PII:
S1381-1177(14)00046-0
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2014.02.002
MOLCAB 2884
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
21-12-2013
6-2-2014
6-2-2014
Please cite this article as: Ingrid C.R. CostaFelipe K. SutiliGabriela V.V. da
SilvaSelma G.F. LeiteLeandro S.M. MirandaRodrigo O.M.A. de Souza Lipase
Catalyzed Ascorbyl Palmitate Synthesis under Microwave Irradiation (2014),
http://dx.doi.org/10.1016/j.molcatb.2014.02.002
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Lipase Catalyzed Ascorbyl Palmitate Synthesis under
Microwave Irradiation
Ingrid C. R. Costa,a,b Felipe K. Sutili, a,b Gabriela V. V. da Silva, b Selma G. F.
Leite, a Leandro S. M. Miranda b and Rodrigo O. M. A. de Souza*b
a Escola de Química, Universidade Federal do Rio de Janeiro, CEP22941909, Rio de Janeiro, Brazil
and b Laboratório de Biocatálise e Síntese Orgânica, Instituto de Química, Universidade Federal do
Rio de Janeiro, Cidade Universitária, 21941-909 Rio de Janeiro-RJ, Brazil
ABSTRACT
The production of ascorbyl palmitate from palmitic acid and ascorbic acid through the
esterification catalyzed by Novozym 435 was performed under microwave irradiation. The
effects of different reaction parameters were evaluated. The reactions were carried out in organic
solvent (acetone, tert-amyl alcohol, cyclohexane, acetonitrile) and the results shown that tert-
amyl alcohol was the best solvent for this reaction. Parameters as temperature, molar ratio and
enzyme loading were evaluated through a central composite design (CCD). In the optimized
conditions, 15% (w/w in relation of ascorbic acid weight) of enzyme, 70°C and molar ratio 1:5,
71% yield of ascorbyl palmitate could be achieved after 1 hour.
Keywords: microwave irradiation, lipases, ascorbyl palmitate, ascorbic acid, palmitic acid
Page 1 of 19

1. INTRODUCTION
Antioxidants are widely used in food industry and cosmetics to prevent oxidative
reactions. Among the natural antioxidants, ascorbic acid (vitamin C) is the most used in the
stabilization of water-based products. However, ascorbic acid is highly hydrophilic and this
limits its application in products formulated with oil and fat derivatives. One approach to solve
this problem is to use lipophilic derivatives such as ascorbyl fatty acid esters (AFAEs). They
have amphiphilic structures and good miscibility with hydrophobic compounds. These AFAEs
are high-potential antioxidants and surfactants with application in diverse products as food and
cosmetics [1-2]. Among the AFAEs, ascorbyl palmitate (L-Ascorbic acid 6-hexadecanoate) is
the most widely used fat-soluble derivative of ascorbic acid. This ester is considered an
innocuous compound and is recognized as safe additive, accepted for human and animal
consumption, usually without specific limitations and restrictions [3].
The synthesis of ascorbyl palmitate is well known [4-9]. Lipase-mediated process
provides an attractive alternative to chemical esterification. Lipases (E.C 3.1.1.3) are the group
of enzymes most used in biotechnology [10]. They are known for their high chemo-,regio- and
stereoselectivity and their ability to catalyze reactions under mild conditions [11]. Although
many authors have already described lipase-catalyzed synthesis of ascorbyl esters, long reaction
times, bio-incompatible solvents, and high price of the enzyme are still some of the drawbacks
for a commercial process [12-13].
The long reaction times of enzymatic production of AFAEs may be overcame by the use
of microwave irradiation. Microwave irradiation (MW), which has proved to be a clean, fast and
convenient energy source, has been widely used in organic chemistry [14]. Traditionally, organic
synthesis is carried out by conductive heating with an external heat source (e.g., an oil bath).
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This method is comparatively slow and inefficient for transferring energy into a reaction system
as it depends on the thermal conductivity of the media and the temperature of the reaction vessel
is higher than the reaction mixture. In contrast, microwave irradiation produces efficient internal
heating by direct coupling of the microwave energy with the solvent, reagent and catalyst
molecules in the reaction mixture, leading to short reaction times and good yields [15-19].
Just a few examples of the application of microwave irradiation in lipase catalytic AFAEs
production can be found over literature. BRADOO and coworkers [20] conducted the
esterification catalyzed by lyophilized lipases form B. stearothermophilus SB-1 and B. cepacia
RGP-10 under microwave irradiation. In this enzymatic synthesis, different fatty acids (C4:0-
C18:1) reacted with ascorbic acid in hexane in a domestic microwave oven in just 30 s reaction
time and power 1.35kW, leading to the desired product in 55 and 58 % yield, respectively.
Kidwai [21] reported the synthesis of AFAEs, esters with different methyl esters of fatty acids
(lauric, myristic, palmitic and stearic) catalyzed by immobilized lipase from Bulkholderia
multivorans under microwave irradiation. All reactions performed under these conditions were
faster than those described in the literature. The system temperature was 80 ± 2 ° C, measured by
an infra-red thermometer. Yields were in the range of 70-80% and the reaction times are 35-45
minutes, depending on the chain of the ester used.
Both works were performed with domestic microwave ovens and typically these systems
can not accurately determine reaction parameters. There is also a lack of pressure control, the
inability to mix the reaction mixture and the heterogeneous distribution of the microwave power
on the oven cavity [15]. All these factors contribute to the low reproducibility and wrong
interpretation of results. However, we point out the potential of this technique in improving
reaction times in synthesis reactions of ascorbyl fatty acid esters, catalyzed by lipases.
Page 3 of 19

Our study describes for the first time the synthesis of ascorbyl palmitate under
microwave irradiation, using controlled reaction conditions, in a dedicated microwave apparatus.
The synthesis was performed through esterification of ascorbic acid and palmitic acid with
Novozym 435 as catalyst (Scheme 1). The optimization of reaction conditions was carried out by
experimental design.
Scheme 1: Lipase catalyzed synthesis of ascorbyl palmitate
2.MATERIALS AND METHODS
2.1.Material
All the reagents and immobilized enzymes, Novozym 435 (Candida antarcica lipase
immobilized on macroporous polyacrylic resin), were purchased from Sigma-Aldrich. Methanol
was HPLC grade.
2.2.Methods
2.2.1.Drying the Solvent
The method used for drying the solvent used 30% (w / v) of 3Å molecular sieve
relative to the volume of the solvent. First, molecular sieves were activated and then were placed
in contact with the solvent for a period of 24h.
2.2.2.Karl Fischer analysis
The amount of H20 was determined by Karl Fischer. The determination was
performed in Titroline KF titrator, Schott. and Hidranal was used as standard. All glasses used in
this procedure were carefully dried and the sample were manipulated with care to avoid water
contamination.
Page 4 of 19

2.2.3. Synthesis of ascorbyl palmitate catalyzed by lipase
Initial studies were performed based on the literature7. The initial conditions were: 0.2
mmol (36mg) of ascorbic acid, 1.0 mmol (261mg) of palmitic acid, 3mL of solvent, 3.6 mg
Novozym 435, 74 mg active molecular sieve (3Å, beads, 8-12 mesh) and the system temperature
was 55 ° C.
The microwave reactions were carried out in a microwave reactor Monowave 300,
Anton-Paar, equipped with two different temperature sensors: infrared and optical fiber. The
reaction were stirred at 600rpm, with magnetic bar, and the applied power was in an “as fast as
possible” mode offered by the software of Monowave 300, in this mode the equipment applies
full power to achieve the desired temperature. The reactions performed with conventional
heating were carried out in refrigerated incubator Marconi, model 830, with agitation at 200 rpm.
2.2.4.HPLC Analysis of samples
Samples from the reactions were diluted with HPLC grade methanol and stored in amber
vial. All samples were immediately injected to avoid decomposition of the sample.
Ascorbyl esters were analyzed on an Agilent 1200 series HPLC, equipped with an
Eclipse XDB-C18 column (5µm, 4.6 mm, 250mm, Agilent), DAD detector (at 245nm). The
column temperature maintained 25°C. Elution was performed with Methanol/H2O/H3PO4
(95/5/0.1) at a flow-rate of 1.0 mL/min, volume injection 10µL. The composition was calculated
by standard samples of ascorbyl palmitate by external method.
2.2.5. Experimental design
A Central Composite Design (CCD) was used to maximize the yield of ascorbyl
palmitate. The optimization study was carried out with three variables, two levels and with
Page 5 of 19

triplicate of central points, totaling eleven experiments points. The levels of the variables were
chosen based on the literature and preliminary studies. Enzyme loading (E, % w/w in relation of
ascorbic acid weight), Temperature (T, °C) and Molar Ratio ascorbic acid : palmitic acid (R,
AA:AP) were chosen for the independent variables and the yield of ascorbyl palmitate (Y, %)
was used as dependent variable. The factorial model is show by (1), and the variables and the
levels selected for the study were represented in the Table 1.
Y= β0 +β1X1+ β2X2+ β3X3+ β12X1X2+ β13X1X3 + β23X2X3 (1)
Where Y= is an investigated experimental response, in this study was the yield, the indicator variable ; X1 represents factor E
(Enzyme loading); X1=-1 represents the low level of 5% and X1=1 represents the high level of 15%. Similarly, X2 and X3
represent factor T (temperature) and R (molar ratio), respectively. β0 is the overall mean, whileβ1;β2 and β3 are the coefficients for
the main effectsof factors E, T and R , respectively. β12, β13 and β23 are the effect coefficients for the ExT, ExR and TxR
interactions.
Table 1. Variable levels in the factorial design.
2.2.6.Statistical analysis
The experimental designs and results analysis were carried out using the software
Statistica 6.0 (Statsoft, Inc, USA). The significance level was established as 95 % to obtain the
mathematical model and the response surface. The significance of the regression coefficients and
the associated probabilities, p(t), were determined by Student’s test, the model equation
significance was determined by Fisher’s test (F-test). The multiple coefficients of correlation R
and the determination coefficient R2 were calculated to evaluate the performance of the
regression equation [22-23].
3. RESULTS AND DISCUSSION
Initially, studies were performed to select the reaction solvent for ascorbyl palmitate
synthesis. Novozym 435 was chosen to catalyze such reactions to be considered as an effective
Page 6 of 19

biocatalyst in the regioselective synthesis of ascorbyl esters. Furthermore, previous studies have
shown that this enzyme is very stable under microwave irradiation [18].
There are many studies about the influence of solvents on the esterification between
ascorbic acid and palmitic acid catalyzed by lipase [24-26]. Based on these studies, we proposed
to make an evaluation of these solvents under microwave irradiation. Since, the performance of a
reaction catalyzed by a biocatalyst is deeply sensitive to the selection of the solvent, the selection
was done based not only on the solubility of substrates, but also in the ability of the solvent to
keep a good enzymatic activity. Additionally, when working with microwave reactions, we
should consider the ability of the solvent to interact with the MW. This difference in the
interaction can provide different heating profiles, faster or slower, affecting reaction rate.
Based on these considerations, we selected four solvents with different log P and
different microwave absorbance level. The medium absorbing solvent (Acetone, Acetonitrile and
tert-amyl alcohol) and the low absorbing or transparent solvent (cyclohexane) were selected. The
reactions were performed with different solvents, under microwave irradiation for 15 minutes
under Novozym 435 catalysis. Unfortunately, we could observe conversions only for the
reactions performed in acetone and tert-amyl alcohol, 2 and 10% respectively.
After this initial results we decided to go ahead with a central composite design 23 in
order to identify important parameters to the synthesis of ascorbyl palmitate catalyzed by
Novozym 435 under microwave irradiation, using tert-amyl alcohol as the solvent. We decided
for the experimental design with three levels because preliminary studies showed that values
above and below the variable values used, not show improvements in the results. For example,
values less than 5% of enzyme loading resulted in low conversions and values above 15%
enzyme loading, will hold a big impact on the final cost of the process. Lower temperatures also
Page 7 of 19

provide lower conversion and higher temperatures than 70 ° C, affect the stability of the enzyme.
Molar ratio, like 1:1, showed low conversions and 1:10 also showed a decrease in performance
when compared to the ratio 1:5, probably by a substrate inhibition. Furthermore, information in
the literature also confirm these limits [7, 8, 17, 18].
The CCD was applied to reduce the number of experiments without compromising the
results. Table 2 shows the matrix with code values, real values and the yields obtained. The first
eight entries are sufficient for determining the mathematical model and refer to the full statistical
design of experiment 23 and the entries 9-11 are the triplicates of the central points, used to
obtaining experimental error.
Table 2. Experimental matrix, with code and real values, and experimental results.
From the results presented in Table 2, it is possible to observe that the highest conversion
could be recorded under the following experimental conditions: enzyme loading 15%,
temperature 70°C, molar ratio (AA:PA) 1:5 and reaction time 15 min (Entry 8, Table 2). The
experimental data have been adjusted to the proposed model (equation 2) and the adequacy of
the model was performed by analysis of variance and parameter R2.
Model:
Y=10.6 + 2.79E + 5.52R + 1.94ET + 1.3ER (2)
Where Y is the percentage yield, and T, R, and E, are the coded values of temperature, molar ratio and enzyme loading,
respectively.
The regression equation was evaluated by the coefficient of correlation (R) and the
determination coefficient (R2). The above model is adequate predictor of the experimental results
as the value of R (0.93) indicates a good agreement between the experimental and predicted
values. Statistical testing of the models was done by the Fisher’s test for ANOVA (Table 3). The
Page 8 of 19

F calculated value was highly significant and was larger than their F tabulated value. According
to the corresponding analysis of variance (ANOVA) of the regression model the F-test for the
regression was significant at a level of 5% (p < 0.05).
Table 3. Variance analysis for the validation of mathematical model (ANOVA)*
The standardized effects of the independent variables and their interactions on the
dependent variable were investigated by preparing the Pareto chart (Fig. 1). The positive
coefficients for the model components (R, E, ExT, ExR, and T) showed a favorable effect on
lipase production of ascorbyl palmitate, while the negative coefficient for the model component
(TxR) indicated an unfavorable effect on the ester production.
Figure 1. Pareto chart from CCD showing the significance of experimental variables: E, enzyme
loading, T, temperature; R, molar ratio.
The independent variable R (molar ratio) has shown the most significant effect in the
studied system. In these experiments the ascorbic acid was used as the limiting reagent, as an
expensive reagent, its uses in excess is not economically attractive. However, excess of palmitic
acid allows the displacement of the reaction in order to product formation, and this reagent can
be recovered after completion of the reaction. The interaction effect between enzyme loading and
temperature parameters also had a significant and positive effect on ascorbyl ester synthesis. In
the experiments, at constant molar ratio, the conversion of ascorbyl palmitate increases with
increasing temperatures and enzyme loading. However, increasing temperatures are limited due
to thermal denaturation of the enzyme. Thus, the Pareto chart indicates that the temperature
variable contributes little to the biosynthesis of ascorbyl palmitate.
Page 9 of 19

As can be seen by the fitted response surface and profiles graphs (Figure 2a and 2b,
respectively) the best conversions could be obtained at high molar ratio and enzyme loading. The
positive effect can be seen also in the graphs. The good correlation between these results
confirmed that the response model was adequate for reflecting the expected optimization. The
evaluation showed that the parameter "curvature check", was not significant and therefore the
linear model is suitable to describe the reaction system and the Equation 2 is satisfactory and
accurate.
Figure 2. Response surface (a) and fitted surface (b) to the esterification reaction in function of
enzyme loading and the molar ratio.
In order to achieve higher yields, we decided to perform the reaction at different reaction
times and follow the conversion to the desired product. As shown in Table 4, increasing reaction
time until 60 minutes is possible to increase the yield towards the desired ascorbyl palmitate to
71%. The same reaction profile under conventional heating does not achieve 40% of conversion
after 24 hours of reaction.
Regarding the increase on yield after 30 minutes, this is still under investigation in our
lab, since, we are interest on side products of oxidation of both product and starting material.
Besides that, this difference can be also related to the gradual solubilization of ascorbic acid
during the reaction course.
The better result obtained under microwave irradiation can be explained by preventing
side reactions such as hydrolysis of fatty acid esters [20-21] and also, to minimize decomposition
of ascorbic acid, highly unstable, due to the short reaction time.
Table 4. Biocatalyzed synthesis of ascorbyl palmitate under microwave irradiation
Page 10 of 19

4. CONCLUSIONS
The synthesis of ascorbyl palmitate using Novozym 435 under microwave irradiation was
investigated using a central composite design. The results showed that the microwave irradiation
provided controlled and efficient heating of our reaction medium. The solvent tert-amyl alcohol
was the best solvent in the ester production. It was show, in the CCD, that the most important
variables were the molar ratio and enzyme loading, both affecting positively the reaction yield.
The variable temperature did not have a significant effect on this synthesis. After the
optimization of the three independent variables (E, T and R), the reaction time was evaluated.
Ascorbyl palmitate yield of 71% was obtained in the optimal conditions: tert-amyl alcohol as
solvent, enzyme loading 15% w/w in relation of ascorbic acid weight, temperature 70°C, molar
ratio (AA:PA) 1:5 and reaction time 60 min.
5. ACKNOWLEDGEMENTS
We thanks CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior); CNPq
(Conselho Nacional de Desenvolvimento Científico e Tecnológico), FAPERJ (Fundação Carlos
Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro) and FINEP (Agência
Financiadora de Estudos e Projetos) for financial support.
6. REFERENCES
[1] S. K. Karmee, Biofuels, Bioprod. Bioref. 2 (2008) 144-154.
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[3] F. A. Andersen, International Journal of Toxicology 18 (1999) 1-26.
[4] T. Stamm, Manufacture of ascorbic acid esters, Patent No. WO 3097626 A1 (2003) 1-15.
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[5] B. Wen, W. Eli, Q. Xue, X. Dong, W. Liu, Ultrasonics Sonochemistry 14 (2007) 213-218.
[6] L. A. Lerin, M.C Feiten, A. Richetti, G. Toniazzo, H. Treichel, M. Mazutti, J. V. Oliveira, E.
G. Oestreicher, D. Oliveira, Ultrasonics Sonochemistry, 18 (2011) 988-996.
[7] C. Humeau, M. Girardin, D. Coulon, A. Miclo, Biotechnology Letters 17 (1995) 1091-1094.
[8] C. Humeau, M. Girardin, B. Rovel, A. Miclo, J. Mol. Catal. B: Enzimatic, 5 (1998) 19-23.
[9] S. K. Karmee, Lipid Technology 23 (2011) 227-229.
[10] M. Kapoor, M. N. Gupta, Process Biochemistry 47 (2012) 55-569.
[11] P. Villeneuve, Biotechnology Advances 25 (2007) 515-536.
[12] H. J. Hsieh, G. R. Nair,W. T. Wu, J. Agric. Food Chem. 54 (2006) 5777-5781.
[13] S. Bradoo, R. K.. Saxena, R. Gupta, J. Am. Oil Chem. Soc. 76 (1999); 1291-1295.
[14] G. D Yadav, P. S. Lathi, J. Mol. Catal. A: Chemical 223 (2004) 51-58.
[15] C.O. Kappe, D. Dallinger, S. S. Muphree, Pratical Microwave Synthesis for Organic
Chemists – Strategies, Instruments an Protocols, Wiley – VCH Verlang GmbH & Co. KGaA,
Weinheim, Germany, 2009.
[16] N.E. Leadbeater, Microwave Heating As A Tool For Sustainable Chemistry; CRC
Press,Florida, US 2010.
[17] R. O. M. A. de Souza, O. A. C. Antunes, W. Kroutil, C. O: Kappe, J. Org. Chem. 74 (2009)
6157-6162.
[18] I. C. R.Costa, S. G. F Leite, I. C. R. Leal, L. S. M. Miranda, R. O. M. A. de Souza, J. Braz.
Chem. Soc. 22 (2011) 1993-1998.
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[19] B. Rejasse, S. Lamare, M. D. Legoy, T. Besson, Journal of Enzyme Inhibition and
Medicinal Chemistry 22 (2007) 518-526.
[20] S. Bradoo, P. Rathi, R. K. Saxena, R Gupta, J. Biochem. Biophys.Methods 51 (2002) 115-
120.
[21] M. Kidwai, P. Mothsra, N. Gupta, S. S. Kumar, R. Gupta, Synthetic Communications 39
(2009) 1143-1151.
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1ºed. São Paulo, Brazil, 2005.
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Page 13 of 19

Table 1. Variable levels in the factorial design.
Values
Variable
Low (-1)
Center (0)
10
High (+1)
E=Enzyme Loading (%)a
T=Temperature (°C)
R=Molar Ratiob
5
15
70
55
1:2
62,5
1:3,5
1:5
a Enzyme loading (% w/w in relation of ascorbic acid weight); b molar ratio (ascorbic acid : palmitic acid).
Table 2. Experimental matrix, with code and real values, and experimental results.
Enzyme
Loading (%)*1
Temperature
(°C)
Molar Ratio
(AA:PA)
Entry
Yield (%)*2
1
2
-1(5)
1(15)
-1(5)
1(15)
-1(5)
1(15)
-1(5)
1(15)
0(10)
0(10)
-1(55)
-1(55)
1(70)
-1 (1:2)
-1(1:2)
-1(1:2)
-1(1:2)
1(1:5)
3,74
4,84
3,85
8,72
15,09
17,42
9,36
23,45
9,50
3
4
1(70)
5
-1(55)
-1(55)
1(70)
6
1(1:5)
7
1(1:5)
8
1(70)
1(1:5)
9
0(62,5)
0(62,5)
0(1:3,5)
0(1:3,5)
10
9,81
Page 14 of 19

11
0(10)
0(62,5)
0(1:3,5)
10,82
*1w/w in relation of ascorbic acid weight.*2The yield was determinate by LC. The reactions were performed in the following
condition: 15 min, 0.2 mmol (36mg) of ascorbic acid, 74mg molecular sieve, 3mL tert-amyl alcohol with 0.05% H2O
(determined by Karl Fischer assay). The other parameters were varied according to the CCD.
Table 3. Variance analysis for the validation of mathematical model (ANOVA)*
Sumof Degrees of
Squares freedom
Mean
Square
Factor
F calculated Ftabulated p-value
2.3x10-4
Regression
350.34
4
87.58
2.37
36.97
4.53
Residuals
Lack of fit
Pure error
TOTAL
14.21
13.26
0.95
6
4
2
364.54
10
*Confidence level 95%.; R2=0,97199; Radj=0,92997
Table 4. Biocatalyzed synthesis of ascorbyl palmitate under microwave irradiation
Entry
Time
15min
30 min
45 min
60 min
Yield (%)a
1
2
3
4
23
25
44
71
aThe yield was determinate by LC
Page 15 of 19

Scheme 1: Lipase catalyzed synthesis of ascorbyl palmitate
Figure 1. Pareto chart from CCD showing the significance of experimental variables: E, enzyme
loading, T, temperature; R, molar ratio.
Page 16 of 19

Figure 2. Response surface (a) and fitted surface (b) to the esterification reaction in function of
enzyme loading and the molar ratio.
Page 17 of 19

Lipase Catalyzed Ascorbyl Palmitate Synthesis under
Microwave Irradiation
a
a
Ingrid C. R. Costa,a,b Felipe K. Sutili a,b
,
Gabriela V. V. da Silva, Selma G. F. Leite,
b
b
Leandro S. M. Miranda and Rodrigo O. M. A. de Souza*
a Escola de Química, Federal University of Rio de Janeiro, CEP22941909, Rio de Janeiro, Brazil
and bLaboratório de Biocatálise e Síntese Orgânica, Instituto de Química, Universidade Federal do
Rio de Janeiro, Cidade Universitária, 21941-909 Rio de Janeiro-RJ, Brazil
Graphical Abstract
Page 18 of 19

Highlights
Æ biocatalyzed synthesis of ascorbyl palmitate
Æ evaluation of microwave irradiation into biocatalyzed process
Æ temperature and time effect on ascorbic acid degradation
Æ experimental design as a tool for optimizing biocatalysis
Page 19 of 19