Optimization of lipase-catalyzed synthesis of cetyl octanoate in supercritical carbon dioxide

Article abstract of DOI:10.1007/s11746-011-1895-8

Cetyl octanoate, a wax ester of 24 carbons, is widely used in the cosmetic industry as a base oil. The current work focuses on lipase-catalyzed synthesis of cetyl octanoate in supercritical carbon dioxide (SC-CO₂) by esterification of cetyl alcohol and octanoic acid. Three immobilized lipases were screened, and 15 reaction conditions were tested in order to find the combination for maximal yield. The results showed that Novozym 435 was the best catalyst for the synthesis, and a reaction time of 20 min was adequate for a maximal yield. Response surface methodology (RSM) with a 3-factor-3-level Box-Behnken design was employed to evaluate the effects of synthesis parameters, including reaction temperature (35-75 °C), pressure (8.27-12.41 MPa), and enzyme amount (5-15% wt of cetyl alcohol). A model for the synthesis was developed and the optimum conditions could be predicted to be reaction pressure of 10.22 MPa, reaction temperature of 63.70 °C, and enzyme amount of 11.20%. An experiment was performed under this optimum condition and a yield of 99.5% was obtained. This experimental yield correlated well with the predicted value of yield (97.6%). We concluded that, in a SC-CO₂ system, nearly 100% molar conversion of cetyl octanoate could be obtained by immobilized Novozym 435 in a short reaction time (20 min) under the predicted optimal conditions.

Full text of DOI:10.1007/s11746-011-1895-8

J Am Oil Chem Soc (2012) 89:103–110
DOI 10.1007/s11746-011-1895-8
ORIGINAL PAPER
Optimization of Lipase-Catalyzed Synthesis of Cetyl Octanoate
in Supercritical Carbon Dioxide
•
Chia-Hung Kuo Hen-Yi Ju Shuan-Wei Chu
•
•
•
Jiann-Hwa Chen Chieh-Ming J. Chang
•
•
Yung-Chuan Liu Chwen-Jen Shieh
Received: 1 November 2010 / Revised: 8 February 2011 / Accepted: 23 March 2011 / Published online: 7 August 2011
Ó AOCS 2011
Abstract Cetyl octanoate, a wax ester of 24 carbons, is
widely used in the cosmetic industry as a base oil. The
current work focuses on lipase-catalyzed synthesis of
cetyl octanoate in supercritical carbon dioxide (SC-CO2)
by esterification of cetyl alcohol and octanoic acid. Three
immobilized lipases were screened, and 15 reaction con-
ditions were tested in order to find the combination for
maximal yield. The results showed that NovozymÒ 435
was the best catalyst for the synthesis, and a reaction
time of 20 min was adequate for a maximal yield.
Response surface methodology (RSM) with a 3-factor-
3-level Box-Behnken design was employed to evaluate
the effects of synthesis parameters, including reaction
temperature (35–75 °C), pressure (8.27–12.41 MPa), and
enzyme amount (5–15% wt of cetyl alcohol). A model for
the synthesis was developed and the optimum conditions
could be predicted to be reaction pressure of 10.22 MPa,
reaction temperature of 63.70 °C, and enzyme amount of
11.20%. An experiment was performed under this opti-
mum condition and a yield of 99.5% was obtained. This
experimental yield correlated well with the predicted
value of yield (97.6%). We concluded that, in a SC-CO2
system, nearly 100% molar conversion of cetyl octanoate
could be obtained by immobilized NovozymÒ 435 in a
short reaction time (20 min) under the predicted optimal
conditions.
Keywords Cetyl octanoate ꢀ Esterification ꢀ
Lipase-catalyzed ꢀ Response surface methodology (RSM) ꢀ
Supercritical carbon dioxide (SC-CO2)
Introduction
Wax esters are long chain esters obtained from natural
sources (e.g. beeswax, sperm whale, skin lipids, jojoba
seeds, carnauba) [1, 2]. Generally, they have carbon chain
lengths of 12 or more. Because of their good emulsifying,
stabilizing, and detergent-like properties, wax esters are
frequently used in industry as carriers of water-insoluble
materials for application onto various surfaces. Wax esters
are biodegradable, biocompatible, nontoxic, and have
excellent wetting capabilities [3]. Wax esters may also be
very useful, particularly in cosmetic and pharmaceutical
applications. Studies have shown that wax esters can be
used as additives or auxiliaries in cleansers, conditioners,
moisturizers, antifoaming agents, lubricants, plasticizers,
polishers, and even anti-arthritis drugs [4, 5].
C.-H. Kuo ꢀ C.-J. Shieh (&)
Biotechnology Center, National Chung Hsing University,
250 Kuo-Kuang Road, Taichung 402, Taiwan
e-mail: cjshieh@nchu.edu.tw
H.-Y. Ju ꢀ S.-W. Chu
Department of Bioindustry Technology, Da-Yeh University,
168 University Road, Chang-Hwa 515, Taiwan
J.-H. Chen
Graduate Institute of Molecular Biology, National Chung Hsing
University, 250 Kuo-Kuang Road, Taichung 402, Taiwan
Cetyl octanoate, a wax ester of 24 carbons, is an analog
of seafowl plumage oil and has an excellent moisture-
retaining capability and non-greasy feeling on skin, making
it an excellent base oil for the cosmetic industry. In
C.-M. J. Chang ꢀ Y.-C. Liu (&)
Department of Chemical Engineering, National Chung Hsing
University, 250 Kuo-Kuang Road, Taichung 402, Taiwan
e-mail: ycliu@dragon.nchu.edu.tw
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J Am Oil Chem Soc (2012) 89:103–110
general, cetyl octanoate is synthesized by esterification of
octanoic acid and cetyl alcohol. Synthesis of cetyl octa-
noate and other wax esters by chemical reaction requires
high reaction temperatures ([100 °C), high pressure, long
reaction time and catalyst (tin, titanium, ferric chloride, or
sulphuric acid) [6–8]. The high temperature and long
reaction time lead to degradation of the ester, and it also has
a high energy cost. Compared with chemical synthesis,
enzymatic synthesis usually takes place at atmospheric
pressure, moderate pH, and moderate temperature [2, 9, 10].
In addition, the enzyme itself is environmentally friendly,
non-toxic, and biodegradable. Wax esters produced by
enzymatic reaction are considered identical to natural wax
esters and therefore can be used safely in cosmetic and food
industries. Lipase-catalyzed synthesis of wax esters,
including oleyl oleate, cetyl myristate, cetyl palmitate, cetyl
oleate, cetyl stearate, oleyl palmitate, and conjugated
linoleic acid (CLA) C6–C22 alkyl esters, have been reported
[2, 5, 10, 11].
Materials and Methods
Materials
Immobilized lipase NovozymÒ 435 (10,000 PLU/g; Propyl
laurate units) from Candida antarctica B (EC 3.1.1.3)
supported on a macroporous acrylic resin, and LipozymeÒ
RMIM (5–6 BAUN/g; Batch acidolysis unit Novo) and
Lipase IM-77 (7.7 BAUN/g) from Rhizomucor miehei (EC
3.1.1.3) supported on a macroporous weak anionic resin
were purchased from Novo Nordisk Bioindustrials Inc.
(Copenhagen, Denmark). Octanoic acid (99% purity), cetyl
alcohol (99% purity) and n-hexane was purchased from
Sigma Chemical Co. (St Louis, MO, USA). Molecular
˚
sieve 4 A was purchased from Davison Chemical (Balti-
more, MD, USA). All other chemicals were purchased
from Sigma-Aldrich (St. Louis, MO) and of analytical
reagent grade.
There is, however, still a major drawback with the
lipase-catalyzed synthesis of wax esters [2, 5, 10, 11].
Most of the syntheses were carried out in an organic
solvent system, causing problems of residual solvent,
environmental pollution, and biohazards. The problem
can be well solved by using supercritical carbon dioxide
(SC-CO2) as the solvent system for the synthesis. Several
detailed review papers on enzymatic reactions in super-
critical fluids (SCF) have been reported and published
[12, 13]. Enzymatic synthesis of wax esters in SC-CO2 is
organic solvent-free, with the extra advantages of easy
separation of unreacted substances, high diffusivity, low
viscosity, and low surface tension [14, 15]. There have
been a few studies regarding lipase-catalyzed synthesis of
wax esters in SC-CO2. This includes synthesis of lauryl
oleate [14], n-octyl oleate [16], and citronellol laurate
[17] in SC-CO2. So far, there has been no reported study
regarding lipase-catalyzed synthesis of cetyl octanoate in
SC-CO2.
Esterification in SC-CO2
Cetyl alcohol and octanoic acid were dehydrated by using a
˚
4-A molecular sieve for 24 h before use. Synthesis of cetyl
octanoate in SC-CO2 was carried out in a high pressure
reactor as depicted in Fig. 1. Carbon dioxide was pres-
surized to the critical point ([7.38 MPa) by a compressor
(Taiwan Supercritical Technology Co., Ltd., Taiwan) and
the reaction temperature was controlled by a LE-529
incubator (Get Bio Co., Taiwan). As is typical for esteri-
fication reactions, the 0.86 ml of reaction mixture con-
tained 30 mM cetyl alcohol, 60 mM octanoic acid, and
various amounts of immobilized lipase (NovozymÒ 435,
Response surface methodology (RSM) is a collection of
mathematical and statistical techniques for designing
experiments, building models, evaluating the relative sig-
nificance of several independent variables, and determining
the optimum conditions for desired responses. It is a useful
tool for understanding the interactions among various
parameters that affect the process and has been applied
successfully for optimizing parameters in various processes
[18, 19]. In this study, different reaction conditions for
lipase-catalyzed synthesis of cetyl octanoate in SC-CO2 are
evaluated. RSM is employed to find out the relationships
between condition factors (pressure, reaction temperature
and enzyme amount) and the response results (molar con-
version), in order to determine the optimal conditions for
synthesis.
Fig. 1 Schematic diagram of the SC-CO2 device. 1, magnetic stirrer;
2, reactor; 3, incubator (temperature controlled); 4, compressor; 5,
pressure gauge; 6, decompression valve
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J Am Oil Chem Soc (2012) 89:103–110
105
Table 1 Box-Behnken design
Treatment no.a
Factor
Experimental molar
conversion (%)
and observed experimental data
for 3-level–3-factor response
surface analysis
X1
pressure (MPa)
X2
temperature (°C)
X3
enzyme amount (%)
1
-1b (8.27)
1 (12.41)
-1 (8.27)
1 (12.41)
-1 (8.27)
1 (12.41)
-1 (8.27)
1 (12.41)
0 (10.34)
0 (10.34)
0 (10.34)
0 (10.34)
0 (10.34)
0 (10.34)
0 (10.34)
-1 (35)
-1 (35)
1 (75)
1 (75)
0 (55)
0 (55)
0 (55)
0 (55)
-1 (35)
1 (75)
-1 (35)
1 (75)
0 (55)
0 (55)
0 (55)
0 (10)
0 (10)
0 (10)
0 (10)
-1 (5)
-1 (5)
1 (15)
1 (15)
-1 (5)
-1 (5)
1 (15)
1 (15)
0 (10)
0 (10)
0 (10)
42.73 0.67
41.66 2.05
98.41 1.01
97.29 0.82
83.93 1.69
78.01 1.46
96.89 1.69
93.82 0.19
29.54 1.37
92.19 0.63
57.13 0.59
98.20 0.33
88.41 1.25
86.25 1.81
89.29 0.51
2
3
4
5
6
7
8
9
10
11
12
13
14
15
a
The treatments were run as a
random order
b
The values -1, 0, and 1 are
coded levels
LipozymeÒ RMIM, or Lipase IM-77). Experiments were
conducted at different temperatures and pressures in the
50 ml reactor for 20 min. All reactions were carried out in
duplicate.
the reaction mixture through glass wool and an anhydrous
sodium-sulfate column. Cetyl octanoate in the reaction
mixture was quantitated by injecting a 1-lL aliquot of the
reaction mixture into a gas chromatograph (GC) (Acme
6100, Young Ling Instrument, Korea) equipped with a
flame-ionization detector (FID) and MTX-65TG fused-
silica capillary column (30 m 9 0.25 mm i.d.; film thick-
ness 0.1 lm; RESTEK, USA) in splitless mode. Injector
and FID temperatures were set at 240 and 250 °C,
respectively, and the oven temperature was maintained at
125 °C for 5 min followed by elevation to 230 °C at a rate
of 50 °C min-1, and then at 230 °C for 12.9 min. Nitrogen
was used as the carrier gas. The molar conversion was
defined as (mmol cetyl octanoate/mmol of initial cetyl
alcohol) 9 100%. Calibration curves were prepared from
cetyl octanoate (chemical synthesis) and cetyl alcohol
standards and samples diluted to the appropriate level to
fall within the calibration curve.
Experimental Design and Statistical Analysis
For synthesis of cetyl octanoate in SC-CO2, a 3-level-
3-factor Box-Behnken design with three replicates at the
center was employed, which required 15 experiments in
total. The variables included pressure (8.27, 10.34, and
12.41 MPa), reaction temperature (35, 55, and 75 °C), and
amount of NovozymÒ 435 (5, 10, and 15% wt of cetyl
alcohol). The experimental data (Table 1) was analyzed by
SAS software. Response surface regression (RSREG) was
used in order to fit the following second-order polynomial
equation [20]:
3
3
2
3
X
X
X X
Y ¼ bk0
þ
bkiXiþ
bkiiXi2þ
bkijXiXj
ð1Þ
i¼1
i¼1
i¼1 j¼iþ1
Results and Discussion
where Y is response (production rate), bk0, bki, bkii and bkij
are constant coefficients, and Xi and Xj the uncoded inde-
pendent variables. The ridge max option was employed to
compute the estimated ridge of maximum response for
increasing the radius from the center of the original design.
Efficacy of Different Catalysts
Three different lipases, NovozymÒ 435, LipozymeÒ RMIM,
and Lipase IM-77, were screened initially for their efficacy
on the synthesis of cetyl octanoate. The reaction was per-
formed at 55 °C with a substrate molar ratio of 1:2 (cetyl
alcohol: octanoic acid) and 10% of enzyme amount (by
weight of cetyl alcohol) in SC-CO2 for 20 min. Fig. 2 shows
results of conversion of cetyl octanoate for the three immo-
bilized lipase at the different pressure. Of the three lipases
Quantitation of Cetyl Octanoate
After the reaction over the desired time, the reaction
mixture was depressurized and eluted with hexane. The
enzyme and any residual water were removed by passing
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J Am Oil Chem Soc (2012) 89:103–110
Fig. 3 The effect of temperature on synthesis of cetyl octanoate in
SC-CO2. The reaction conditions were set as: substrate molar ratio,
1:2 (alcohol:fatty acid); NovozymÒ 435 amount, 10%; agitation
speed, 100 rpm; reaction time, 20 min; pressure, 10.34 MPa; and
reaction temperature, 35–75 °C
Fig. 2 Screening of different catalysts for synthesis of cetyl octano-
ate in SC-CO2. The reaction conditions were set as: substrate molar
ratio, 1:2 (alcohol:fatty acid); enzyme amount, 10%; agitation speed,
100 rpm; reaction temperature, 55 °C; reaction time, 20 min; and
pressure, 8.27–19.31 MPa
tested, NovozymÒ 435 showed the best activity with maxi-
mum conversion of cetyl octanoate and Lipase IM77 the
least. At a reaction pressure of 10.34 MPa, approximately
90% molar conversion of cetyl octanoate was achieved with
NovozymÒ 435. In other words, this experiment demon-
strated a high yield of cetyl octanoate in SC-CO2 could be
achieved within a short time (20 min) via NovozymÒ 435
catalysis. In other studies, Novozym 435 has also been
shown to be very active and useful in catalyzing the synthesis
of esters [21–24]. For this reason, NovozymÒ 435 was
employed for the following experiments in this study.
and that activity increased as the temperature increased
(Fig. 3). From these results, it appeared that deformation of
NovozymÒ 435 at temperatures higher than 60 °C probably
would not occur in SC-CO2.
Model Fitting
One aim of this study was to develop a statistical model for
understanding the relationships between the manipulated
variables and the response (molar conversion of cetyl
octanoate) in the NovozymÒ 435-catalyzed synthesis of
cetyl octanoate in SC-CO2. Compared with one-factor-at-a-
time design used in most of the studies found in the liter-
ature, RSM is less frequently used and yet is very efficient
in reducing the number of experimental treatments and
times to obtain optimal reaction conditions. The experi-
mental conditions and the response values from the exper-
imental Box-Behnken design are listed in Table 1, which
shows that the highest molar conversion (98.20 0.33%)
was obtained at 10.34 MPa, 75 °C, and 15% enzyme
(treatment no. 12), and the lowest (29.54 1.37%) was
achieved at 10.34 MPa, 35 °C, and 5% enzyme (treatment
no. 9). From the SAS output of RSREG, the second-order
polynomial Eq. (2) was given as below:
Effect of Reaction Temperature
The effect of temperature on cetyl octanoate synthesis in
SC-CO2 was tested in the range of 35–75 °C and the results
are shown in Fig. 3. The conversion increased with
increasing temperature. The highest conversion was found
from 65 to 75 °C where at least 90% conversion was
achieved. All further experiments using NovozymÒ 435
were performed below 75 °C. According to its product
sheet, NovozymÒ 435 is a heat-tolerant immobilized
enzyme with maximum activity at 70–80 °C. However, on
the product sheet, it is suggested that the enzyme be used at
40–60 °C for stability purposes. It was previously reported
that enzymatic synthesis of sugar fatty acid esters in
2-methyl-2-butanol solvent at temperatures higher than
60 °C would deform the enzyme with a decrease in activity,
but in SC-CO2 the optimal reaction temperature was shifted
to 80 °C. The increased stability of lipase in SC-CO2 could
be due to the rigidity of the immobilized lipase in SC-CO2
at high temperatures [25]. Our results also clearly indicated
that NovozymÒ 435 was active in SC-CO2 from 65 to 75 °C
Yð%Þ ¼ ꢁ146:112593 ꢁ 3:607214x1 þ 6:954580x2
þ 4:046022x3 þ 0:109299x1x1 ꢁ 0:000302x2x1
ꢁ 0:046073x2x2 þ 0:068841x3x1 ꢁ 0:053950x3x2
ꢁ 0:011567x3x3
ð2Þ
The plot of experimental values of the conversion (%)
versus those calculated from Eq. (2) indicates a good fit, as
shown in Fig. 4. The analysis of variance (ANOVA),
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J Am Oil Chem Soc (2012) 89:103–110
107
summarized in Table 2, reveals that this second-order
polynomial model, having a very small P-value (\0.0001)
and high coefficient of determination (R2 = 0.9973), was
highly significant and adequate to represent the actual
relationship between the response (molar conversion) and
the independent variables. Furthermore, the overall effects
of the three synthesis variables of the molar conversion
were analyzed and reported in Table 3. The P-values mark
the significance of coefficients and are also important for
understanding the pattern of the mutual interactions
between the parameters. A value of Prob. [ F \ 0.05
indicates that the model terms are significant. Table 3
demonstrates that the linear term of the temperature (x2;
P \ 0.0001) and the enzyme amounts (x3; P = 0.0001) are
significant parameters for cetyl octanoate production. The
quadratic (x2 9 x2; P \ 0.0001) and interactions (x2 9 x3;
P = 0.0032) terms are also statistically significant. How-
ever, the variable pressure and its quadratic or interactions
terms did not display any significant effect. In this study,
where pressures from 8.27 to 12.41 MPa were studied,
pressure was found to not strongly influence the product
yield. This is somewhat unexpected since pressures of 9–14
MPa correspond to log P (index of the polarity of solvent)
of 1.536–2.74 in this system, which is postulated to be the
best region for the esterification reaction. However, a
similar result was also reported in a study of lavandulol
esterification [26]. On the other hand, Knez et al. and
Laudani et al. [14, 16] demonstrated the highest degree of
wax ester synthesis is at 10 MPa, and that if the pressure
was out of the range 8.27–12.41 MPa the degree of ester-
ification significantly decreased.
Mutual Effect of Parameters
The relationships between reaction factors and responses
can be better understood by examining the series of response
surface plots generated by holding constant the enzyme
amount (Fig. 5), reaction temperature (Fig. 6), or pressure
(Fig. 7). Reaction temperature and pressure were investi-
gated in the range of 35–75 °C and 8.27–12.41 MPa,
respectively. Fig. 5 shows the effect of varying temperature
and pressure on esterification with 5% enzyme loading. At
any given pressure from 8.27–12.41 MPa, an increase in
temperature to 75 °C led to a curvilinear increase in molar
conversion to 95%, indicating that the temperature was one
of the most important factors in the synthesis of cetyl octa-
noate. However, the reaction pressure had no significant
effect on the conversion. This result was consistent with that
shown in Table 3, which indicated that reaction pressure (x1)
did not have a significant effect (P [ 0.05) on the synthesis
of cetyl octanoate. However, increasing or decrease the
pressure out of this study range (8.27–12.41 MPa) signifi-
cant decreases cetyl octanoate production as shown in
Fig. 2. The effect of varying enzyme amounts and pressure
on molar conversion at constant reaction temperature
(55 °C) is shown in Fig. 6. At any given pressure, an
increasing enzyme amount seemed to have higher molar
conversion. At the reaction condition of lowest pressure
(8.27 MPa) and highest enzyme amount (15%), the maximal
molar conversion (96.6%) would be reached. However,
increasing pressure resulted in only a slight decrease in
esterification efficiency at any given enzyme amount. This
indicates that NovozymÒ 435 is stable in SC-CO2 within the
experimental pressure range. Fig. 7 shows the effect of
Fig. 4 The relationship between predicted and experimental molar
conversion of cetyl octanoate
Table 2 ANOVA for synthesis
Source
Degrees of freedom
Sum of squares
F value
Prob. [ Fa
variables pertaining to the
response percent molar
conversion
Linear
3
3
3
9
3
2
5
6,281.613775
1,270.931883
118.455350
7,671.001008
15.897125
503.55
101.88
9.50
\0.0001
\0.0001
0.0166
Quadratic
Cross product
Total model
Lack of fit
Pure error
Total error
R2 = 0.9973
204.98
2.17
\0.0001
0.3314
4.893867
20.790992
a
(Prob. [ F) = level of
significance
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J Am Oil Chem Soc (2012) 89:103–110
Table 3 ANOVA for the fitted quadratic polynomial model of all
independent variables
Factor
Degrees of
freedom
Sum of
squares
Prob. [ Fa
Pressure (x1)
Temperature (x2)
Enzyme amount (x3)
X1 9 X2
1
1
1
1
1
1
1
1
1
15.624
0.1103
\0.0001
0.0001
5,779.737
486.252
0.001
0.9907
X1 9 X3
2.031
0.5158
X2 9 X3
116.424
0.810
0.0032
X1 9 X1
0.6774
X2 9 X2
1,254.0339
0.309
\0.0001
0.7961
X3 9 X3
Underlined model terms are significant ones
a
(Prob. [ F) = level of significance
Fig. 6 Response surface plot showing the mutual effect of Nov-
ozymÒ 435 amount and reaction pressure on molar conversion of
cetyl octanoate. The reaction temperature was kept constant (55 °C)
throughout the experiment
Fig. 5 Response surface plot showing the mutual effect of reaction
temperature and pressure on molar conversion of cetyl octanoate. The
NovozymÒ 435 amount was kept constant (5%) throughout the
experiment
Fig. 7 Response surface plot showing the mutual effect of Nov-
ozymÒ 435 amount and reaction temperature on molar conversion of
cetyl octanoate. The reaction pressure was kept constant (8.27 MPa)
throughout the experiment
varying enzyme amount and temperature on esterification at
constant pressure (8.27 MPa). With the highest enzyme
amount (15%) and an appropriate reaction temperature
(65 °C) condition, the maximum percent molar conversion
of cetyl octanoate was obtained. However, a lower reaction
temperature and enzyme amount significantly decreased the
molar conversion to 30%, indicating that both the enzyme
amount and reaction temperature were the two most
important parameters for the synthesis of cetyl octanoate.
to estimate optimal experimental-variable values at fixed
distances from a defined center of an experimental region
[27]. The radius is the given distance from the ridge
starting point at which to compute the optima. The ridge
analysis method computes the estimated ridge of maximum
response for increasing radius from the center of original
design. The ridge max analysis (Table 4) predicted maxi-
mum conversion to be 97.55 1.11% at 10.22 MPa,
63.70 °C, and 11.20% enzyme. The validity of the pre-
dicted model was examined by experiments at the
Attaining Optimum Conditions
The optimum point was determined by ridge max analysis
[20]. The ridge analysis uses a quadratic regression model
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J Am Oil Chem Soc (2012) 89:103–110
109
Table 4 Estimated ridge of
maximum response for variable
percent molar conversion
Coded
radius
Pressure
(MPa)
Temperature
(°C)
Enzyme
amount (%)
Estimated
response (%)
Standard
error
0
10.34
10.33
10.32
10.29
10.26
10.22
55.00
56.91
58.77
60.58
62.25
63.70
10.00
10.15
10.32
10.54
10.82
11.20
87.98
90.60
92.86
94.75
96.31
97.55
1.18
1.17
1.16
1.15
1.13
1.11
0.1
0.2
0.3
0.4
0.5
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predicted optimum conditions. The molar conversion of the
actual experiment was 99.47 0.05%. The result indi-
cated that observed value was almost the same as that
predicted from the equation (Eq. 1). The model generated
adequately predicted the percent molar conversion.
Conclusions
Cetyl octanoate, a wax ester of 24 carbons, was synthesized
enzymatically within a short time (20 min) in the green
solvent ‘‘SC-CO2’’. Three enzymes were examined;
Novozyme 435 was chosen for further study since it
demonstrated the highest activity. The optimum reaction
condition for high conversion of cetyl octanoate was
achieved by using Box-Behnken design and response sur-
face methodology. Three parameters, i.e., reaction tem-
perature, pressure, and enzyme amount, were evaluated by
joint test. Based on the results of the evaluation, it appears
that reaction temperature and enzyme amount significantly
affected the molar conversion, but reaction pressure did not
affect yield within the range of pressures studied here,
8.27–12.41 MPa. A model for the synthesis was built and
the optimal synthesis condition was derived as follows:
reaction pressure, 10.22 MPa; reaction temperature,
63.7 °C; and enzyme amount, 11.20%. Under this optimal
condition, a conversion value of 99.5% could be expected.
Synthesis of cetyl octanoate under this optimal condition
was performed experimentally and 97.6% conversion was
obtained. Thus, optimization of the synthesis of cetyl
octanoate catalyzed by NovozymÒ 435 was successfully
developed by Box-Behnken design and RSM.
´
´
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