Optimization of enzymatic synthesis of cetyl 2-ethylhexanoate by Novozym 435

Article abstract of DOI:10.1007/s11746-011-1868-y

Waxes are esters obtained from long-chain fatty acids and long-chain alcohols which are biodegradable, biocompatible and nontoxic. Seafowl feather oil is a natural wax ester that exists on seafowl feathers. Cetyl 2-ethylhexanoate is the major ingredient of seafowl feather oil. Cetyl 2-ethylhexanoate is widely used in cosmetics as a base oil because of its lubricity, moisture retention and non-toxic properties. An optimal production of cetyl 2-ethylhexanoate by direct esterification of cetyl alcohol with 2-ethylhexanoic acid was developed using an immobilized lipase (Novozym 435) as a catalyst in n-hexane. Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) were employed to evaluate the effects of reaction time, reaction temperature, substrate molar ratio, and enzyme amount on the yield of cetyl 2-ethylhexanoate. The results show that reaction time, reaction temperature, substrate molar ratio, and enzyme amount have significant effects on the yield of the esterification reaction. On the basis of ridge-max analysis, the optimum conditions were as follows: a reaction time of 2.65 days, a reaction temperature of 56.18 °C, a substrate molar ratio of 2.55:1, and an enzyme amount of 251.39%. The predicted and experimental values of molar conversion were 91.95 and 89.75 ± 1.06%, respectively. AOCS 2011.

Full text of DOI:10.1007/s11746-011-1868-y

J Am Oil Chem Soc (2011) 88:1917–1923
DOI 10.1007/s11746-011-1868-y
ORIGINAL PAPER
Optimization of Enzymatic Synthesis of Cetyl 2-Ethylhexanoate
by NovozymÒ 435
•
Hsiao Ching Chen Chia-Hung Kuo
•
•
Hsin Hung Chen Yung-Chuan Liu
•
Chwen Jen Shieh
Received: 3 January 2011 / Revised: 21 April 2011 / Accepted: 26 May 2011 / Published online: 18 June 2011
Ó AOCS 2011
Abstract Waxes are esters obtained from long-chain
fatty acids and long-chain alcohols which are biodegrad-
able, biocompatible and nontoxic. Seafowl feather oil is a
natural wax ester that exists on seafowl feathers. Cetyl
2-ethylhexanoate is the major ingredient of seafowl feather
oil. Cetyl 2-ethylhexanoate is widely used in cosmetics as a
base oil because of its lubricity, moisture retention and
non-toxic properties. An optimal production of cetyl
2-ethylhexanoate by direct esterification of cetyl alcohol
with 2-ethylhexanoic acid was developed using an immo-
bilized lipase (NovozymÒ 435) as a catalyst in n-hexane.
Response surface methodology (RSM) and 5-level-4-factor
central composite rotatable design (CCRD) were employed
to evaluate the effects of reaction time, reaction tempera-
ture, substrate molar ratio, and enzyme amount on the yield
of cetyl 2-ethylhexanoate. The results show that reaction
time, reaction temperature, substrate molar ratio, and
enzyme amount have significant effects on the yield of the
esterification reaction. On the basis of ridge-max analysis,
the optimum conditions were as follows: a reaction time of
2.65 days, a reaction temperature of 56.18 °C, a substrate
molar ratio of 2.55:1, and an enzyme amount of 251.39%.
The predicted and experimental values of molar conversion
were 91.95 and 89.75 1.06%, respectively.
Keywords Biocatalysis ꢀ Cetyl 2-ethylhexanoate ꢀ
Lipase ꢀ Optimization ꢀ Response surface methodology ꢀ
Wax esters
Introduction
Wax esters are esters that are derived from long-chain fatty
acids and long-chain fatty alcohols. They are natural
chemicals and can be extracted from jojoba oil, sperm
whale oil, beeswax, and seafowl feathers [1, 2]. The
presence of wax esters on feathers provides the water-
proofing properties of the plumage so that seafowl can
swim, dive and rest on the water. Cetyl 2-ethylhexanoate is
one of the main wax esters found on seafowl feathers, and
its chemical structure is shown in Scheme 1.
The compounds are formulated in numerous personal
care products due to their excellent wetting behavior at
interfaces and non-greasy feeling when applied on the skin
surface [3]. They are also important ingredients in cos-
metics, pharmaceuticals, lubricants, plasticizers, and pol-
ishes [1, 4].
Since the cost and availability of wax esters from natural
resources such as jojoba oil and sperm whale oil are lim-
ited, the attempts to synthesize wax esters with cheaper
starting materials and simple methods have become very
important [1]. Wax esters can be synthesized by chemical
or enzymatic reactions. Compared to chemical catalysis,
lipase-catalyzed synthesis is more substrate specific and
therefore suitable for producing high-quality natural prod-
ucts. The enzymatic reaction is conducted under moderate
reaction conditions (pH, temperature, atmospheric
H. C. Chen ꢀ H. H. Chen
Department and Graduate Program of Bioindustry Technology,
Dayeh University, Chang-Hua 515, Taiwan
C.-H. Kuo ꢀ C. J. Shieh (&)
Biotechnology Center, National Chung Hsing University,
Taichung 402, Taiwan
e-mail: cjshieh@nchu.edu.tw
Y.-C. Liu
Department of Chemical Engineering,
National Chung Hsing University, Taichung 402, Taiwan
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J Am Oil Chem Soc (2011) 88:1917–1923
Scheme 1 Lipase-catalyzed
synthesis of cetyl
Lipase
2-ethylhexanoate from cetyl
alcohol and 2-ethylhexanoic
acid
+
+
H2O
n-hexane
2-Ethylhexanoic acid
Cetyl2-ethylhexanoate
Cetyl alcohol
Water
pressure) and requires only simple steps to purify the
product from the reaction mixture. The wax esters obtained
from the enzymatic reaction can be labeled as ‘natural
identical’ and used in cosmetics or food products.
Units)/g containing 1–2% (w/w) water. Cetyl alcohol (99%)
and 2-ethylhexanoic acid (99%) were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). Cetyl 2-ethylhexanoate
was purchased from Kao Chemicals Co. (Tokyo, Japan). A
˚
molecular sieve (4 A) was purchased from Davison Chem-
There are many reports investigating the synthetic
methods for wax esters. Hunter et al. [5] synthesized cetyl
myristoleate by chemical reactions between myristoleic
acid and cetyl alcohol. Decagny et al. [6] and Hadzir et al.
[7] reported the lipase-catalyzed synthesis of wax esters
through triolein alcoholysis with stearyl/oleyl alcohol. Wax
esters can also be produced by lipase-catalyzed alcoholysis
of vegetable oils with different alcohols [8]. Petersson et al.
[9] successfully prepared cetyl palmitate in a solvent-free
process using an immobilized lipase as catalyst. For the
optimal synthesis of wax esters via lipase catalysis, it is
important to design a simple and efficient enzymatic bio-
synthesis system from large experimental variables and to
obtain the optimum production of valuable products using
an appropriate solvent formulation over a short time period
and with minimal trials. Several functional statistical
models, such as response surface methodology (RSM),
have been successfully applied to investigate the possible
interactions and to optimize various valuable wax ester
production by lipase [1, 3, 8]. So far, there have been no
reports regarding lipase-catalyzed synthesis of cetyl
2-ethylhexanoate. The literature is limited on lipase-cata-
lyzed esterification from ethyl branched acids and long
chain alcohol substrates.
ical (Baltimore, MD, USA), and n-hexane was obtained from
the Merck Chemical Co. (Darmstadt, Germany). All of the
other chemicals were of analytical reagent grade.
Experimental Design
A 5-level-4-factor CCRD was employed in this study; 27
experiments were run. The fractional factorial design
consisted of 16 factorial points, 8 axial points, and 3 center
points. The variables and their levels were: reaction time
(1–3 days), reaction temperature (45–65 °C), substrate
molar ratio (2-ethylhexanoic acid:cetyl alcohol = 1–3:1,
w/w), and enzyme amount (NovozymÒ 435/cetyl alco-
hol = 100–300%, w/w). Table 1 shows the independent
factors (xi), levels and experimental design, both coded and
uncoded. The 27 runs were performed in a fully random
order to avoid bias.
Enzymatic Esterification
All of the reagents were dehydrated by molecular sieves
˚
(4 A) for 24 h. Lipase-catalyzed synthesis of cetyl 2-eth-
ylhexanoate from cetyl alcohol and 2-ethylhexanoic acid is
presented in Scheme 1. Cetyl alcohol (0.1 M), different
molar ratios of 2-ethylhexanoic acid and different amounts
of NovozymÒ 435 were well mixed in n-hexane. The
esterification reaction was carried out in an orbital shaking
water bath (200 rpm) under various reaction temperatures
and reaction times as shown in Table 1.
In this study, an RSM and a 5-level-4-factor central
composite rotatable design (CCRD) were employed to
investigate the affinities between the reaction variables
(reaction time, reaction temperature, substrate molar ratio,
and enzyme amount) and response (yield %), and to obtain
the optimal conditions for lipase-catalyzed synthesis of
wax esters with branched fatty acids.
Determination of Cetyl 2-Ethylhexanoate
Materials and Methods
The cetyl 2-ethylhexanoate formation was determined by
injecting 1 lL of the reaction mixture in splitless mode into
a gas chromatograph (Hewlett Packard 7890, Avondale,
PA, USA) equipped with a flame-ionization detector (FID)
and an MXT-65TG fused silica capillary column
(30 m 9 0.25 mm i.d.; film thickness 1 lm; Restek,
Bellefonte, PA, USA). Injector and FID temperatures were
set at 240 and 250 °C, respectively. The oven temperature
was maintained at 125 °C for 5 min, increased to 230 °C at
Materials
Immobilized lipase (triacylglycerol hydrolase, EC 3.1.1.3;
NovozymÒ 435) from Candida antarctica, supported on
acrylic resin, was purchased from Novo Nordisk Bioin-
dustrials, Inc. (Bagsvaerd, Denmark). The catalytic activity
of NovozymÒ 435 was 10,000 PLU (Propyl Laurate
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J Am Oil Chem Soc (2011) 88:1917–1923
1919
Table 1 Central composite
Treatmenta Factors
Experimental Predicted
yieldd (%)
rotatable design and
experimental data for 5-level-4-
factor and response surface
analysis
yield (%)
Reaction
time (day)
X1
Reaction
temperature
(°C) X2
Substrate molar Enzyme
ratio (Ac:Al)b
X3
amount
(%)c X4
1
-1(1.5)
-1(1.5)
-1(1.5)
-1(1.5)
1(2.5)
1(2.5)
1(2.5)
1(2.5)
-1(1.5)
-1(1.5)
-1(1.5)
-1(1.5)
1(2.5)
1(2.5)
1(2.5)
1(2.5)
-2(1)
2(3)
-1(50)
-1(50)
1(60)
-1(1.5)
1(2.5)
-1(1.5)
1(2.5)
-1(1.5)
1(2.5)
-1(1.5)
1(2.5)
-1(1.5)
1(2.5)
-1(1.5)
1(2.5)
-1(1.5)
1(2.5)
-1(1.5)
1(2.5)
0(2)
1(250)
-1(150)
-1(150)
1(250)
45.42 0.01 42.86
32.50 0.87 33.07
63.47 3.34 58.39
72.84 1.11 74.43
44.68 2.47 41.83
79.79 0.13 83.61
73.97 2.61 72.15
77.10 0.87 78.40
28.28 0.83 23.43
65.52 0.22 59.53
65.43 0.02 62.33
69.80 3.51 63.47
59.54 0.41 61.55
58.08 1.23 56.86
66.25 2.71 67.92
89.11 1.35 89.65
36.12 0.43 46.21
84.33 0.09 79.82
36.18 1.36 38.93
77.10 4.36 79.93
40.32 2.77 45.83
72.89 2.20 72.97
39.57 1.01 45.18
75.90 1.88 75.87
63.67 2.81 63.54
63.59 2.99 63.54
63.35 1.46 63.54
2
3
4
1(60)
5
-1(50)
-1(50)
1(60)
-1(150)
1(250)
6
7
1(250)
8
1(60)
-1(150)
-1(150)
1(250)
9
-1(50)
-1(50)
1(60)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1(250)
1(60)
-1(150)
1(250)
-1(50)
-1(50)
1(60)
-1(150)
-1(150)
1(250)
1(60)
0(55)
0(200)
0(55)
0(2)
0(200)
0(2)
-2(45)
2(65)
0(2)
0(200)
a
All treatments were run in
random order
0(2)
0(2)
0(200)
0(2)
0(55)
-2(1)
2(3)
0(200)
b
(Ac:Al) was the molar ratio
of 2-ethylhexanoic acid:cetyl
alcohol
0(2)
0(55)
0(200)
0(2)
0(55)
0(2)
-2(100)
2(300)
c
0(2)
0(55)
0(2)
The enzyme amount is the
weight percentage of cetyl
alcohol
0(2)
0(55)
0(2)
0(200)
0(2)
0(55)
0(2)
0(200)
d
The predicted yields were
calculated by RSREG equation
0(2)
0(55)
0(2)
0(200)
4
4
3
4
a rate of 50 °C/min, and then kept at 230 °C for 11.9 min.
Nitrogen was used as the carrier gas.
X
X
X X
Y ¼ bk0
þ
bkixi þ
bkiix2i þ
bkijxixj
ð1Þ
i¼1
i¼1
i¼1 j¼iþ1
The yield % was defined as (mmol of cetyl 2-ethyl-
hexanoate/mmol of initial cetyl alcohol) 9 100% and was
estimated using the peak area integrated by on-line soft-
ware Hewlett Packard 6890 Series II Chem Station
(Hewlett Packard 6890, Avondale, PA, USA). A gas
chromatogram of the reaction mixture is shown in Fig. 1.
There are 4 peaks in the GC analysis: cetyl alcohol (sub-
strate), 2-ethylhexanoic acid (substrate), n-hexane (solvent)
and cetyl 2-ethylhexanoate (product). The retention time
was increased in tandem with the carbon number.
where Y is the response (yield %), Bk0, Bki, Bkii and Bkij are
constant coefficients and xi and xj are the uncoded inde-
pendent variables. The ridge-max option was used to
compute the estimated ridge of maximum response for
increasing the radii from the center of the original design.
Results and Discussion
Statistical Analysis
Effects of Reaction Parameters
The experimental data (Table 1) were analyzed by the
response surface regression (RSREG) procedure of SAS
software to fit the following second-order polynomial
equation:
The influence of reaction time on the yield of cetyl 2-ethyl-
hexanoate is shown in Fig. 2; when the reaction time
increased, the initial yield of cetyl 2-ethylhexanoate also
increased. The highest yield (*95%) was obtained from a
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J Am Oil Chem Soc (2011) 88:1917–1923
100
80
60
40
20
0
Fig. 1 Gas chromatogram of the reaction mixture. Each peak was
identified by comparison with corresponding standards
0
1
2
3
4
5
6
Time (Day)
300% enzyme amount after 3 days. After that, the curve
leveled off. In contrast, the yield was only 40% after 5 days at
30% enzyme amount. Many researchers have shown that the
addition of a molecular sieve facilitated the reaction rate of
esterification because of the removal of excess water [10–12].
However,theyieldwasnotsignificantlyincreasedinourstudy
when the molecular sieve was added into the reactionmixture.
From preliminary experimental data in Fig. 2, the variables
and their levels selected in this study were: reaction time
(1–3 days); temperature (45–65 °C); substrate molar ratio
(1–3:1; Ac:Al);and enzymeamount(100–300%), asshown in
Table 1. Table 1 also shows the actual yields obtained from
experiments as well as the predicted yields derived from the
model. Both values were reasonably close, indicating the
effectiveness of the statistical analysis used in this study.
Fig. 2 The effects of reaction time on the yield of cetyl 2-ethylhex-
anoate. Reaction conditions: reaction temperature of 55 °C, substrate
molar ratio of 2:1 (acid:alcohol), open circles enzyme amount of
300%, no molecular sieve; closed circles enzyme amount of 300%,
molecular sieve of 15 mg; open inverted triangles enzyme amount of
30%, no molecular sieve; close inverted triangles enzyme amount of
30%, molecular sieve of 15 mg
Table 2 Analysis of variance for synthesis variables pertaining to the
response of percent yield
Source
Degree of
freedom
Sum of
squares
F value
Pr [ F
Linear
4
6733.6609
40.4515
53.13
0.32
\0.0001a
0.8598
Quadratic
Cross product
Total model
Lack of fit
Pure error
Total error
R2
4
6
418.2584
7192.3709
380.1856
0.0555
2.20
0.1155
\0.0001a
0.0007a
14
16.21
1370.86
Model Fitting
10
2
The RSREG procedure was employed to fit the second-
order polynomial Eq. 1 to the experimental data (Table 1).
Among the various treatments (Table 1), the highest molar
conversion (89.11 1.35%) was treatment no. 16 (a
reaction time of 2.5 days, a reaction temperature of 60 °C,
a substrate molar ratio of 2.5:1, and an enzyme amount of
250%). The lowest molar conversion (28.28 0.83%) was
treatment no. 9 (a reaction time of 1.5 days, a reaction
temperature of 50 °C, a substrate molar ratio of 1.5:1, and
an enzyme amount of 150%). From the SAS output of
RSREG, the second-order polynomial Eq. 2 obtained is
given below (X1: reaction time, X2: temperature, X3: sub-
strate molar ratio and X4: enzyme amount):
12
380.2411
0.9498
a
p \ 0.01, significant at 1% level
polynomial model is an adequate representation of the
actual relationship between the response and the significant
variables, with a very small p value (0.0001) and a
satisfactory coefficient of determination (R2 = 0.9498).
Furthermore, the overall effect of the four synthesis
variables on the yield of cetyl 2-ethylhexanoate was
further analyzed by a joint test (Table 3). These results
revealed that reaction time, reaction temperature, substrate
molar ratio, and enzyme amount are important parameters
Yð%Þ ¼ ꢁ562:99þ56:318X1 þ12:353X2 þ30:377X3
þ 0:980X4 ꢁ 0:522X12 ꢁ 0:887X1X2 ꢁ 0:041X22
þ5:395X1X3 ꢁ 0:456X2X3 ꢁ4:142X32 þ 0:003X1X4
which have
a statistically significant overall effect
(p \ 0.01) on the yield of cetyl 2-ethylhexanoate.
Mutual Effect of Parameters
ꢁ 0:016X2X4 þ0:070X3X4 ꢁ 0:0003X42
ð2Þ
Reaction times and reaction temperatures were investigated
in the range of the reaction time of 1–3 days and
The analysis of variance (ANOVA) data are shown in
Table 2. The results indicate that the second-order
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J Am Oil Chem Soc (2011) 88:1917–1923
1921
temperatures of 45–65 °C, respectively. Fig. 3a represents
the effect of varying reaction time and reaction temperature
on esterification efficiency at a substrate molar ratio of 2:1
and an enzyme amount of 200%. With the highest reaction
temperature (65 °C) and highest reaction time (3 days),
cetyl 2-ethylhexanoate yield of 89% was obtained.
Whereas, when the reaction temperature was decreased to
45 °C and the reaction time shortened (1 day), only 13%
yield remained.
substrate molar ratio of 2:1. At the lowest reaction tem-
perature (45 °C) and enzyme amount (100%), the yield was
5%. The yield increased remarkably when the reaction
temperature and enzyme amount increased. The effect of
substrate molar ratio and reaction temperature on esterifi-
cation efficiency at a constant reaction time (2 days) and a
constant enzyme amount (200%) is shown in Fig. 3c. At
any given substrate molar ratio (1–3:1; Ac:Al), an increase
in reaction temperature tends to give higher yields of cetyl
2-ethylhexanoate. At the highest substrate molar ratio (3:1)
and highest reaction temperature (65 °C), the yield of cetyl
2-ethylhexanoate was 84%.
Figure 3b shows the effects of enzyme amount, reaction
temperature and their mutual interaction on cetyl 2-ethyl-
hexanoate synthesis at a reaction time of 2 days and a
The relationships between reaction factors and response
can be better understood by examining the planned series
of contour plots (Fig. 4) generated from the predicted
model by holding constant enzyme amounts (150, 200,
250%) and substrate molar ratios (1.5:1, 2:1, 2.5:1).
Fig. 4a–c represent the same enzyme amount (150%); and
a, d, and g represent the same substrate molar ratio (1.5:1).
In general, all nine contour plots in Fig. 4 exhibit similar
behavior, in that predicted yield increased according to
increased reaction time and reaction temperature. The
reaction with a 250% enzyme amount and substrate molar
ratio 2.5:1 (Fig. 4i) is suggested as the optimal condition
Table 3 Analysis of variance for the joint test
Factor
Degree of Sum of
freedom
F value Pr [ F
squares
Reaction time (X1)
Temperature (X2)
5
5
5
1803.0113 11.38
2883.7068 18.20
0.0003a
\0.0001a
0.0018a
Substrate molar
ratio (X3)
1226.9909
7.74
Enzyme amount (X4)
5
1714.2826 10.82
0.0004a
a
p \ 0.01, significant at 1% level
Fig. 3 Response surface plots
showing the relationships
between cetyl 2-ethylhexanoate
yield and reaction parameters:
a reaction time and temperature;
b enzyme amount and
(a)
(b)
100
80
100
80
60
40
temperature; c substrate molar
ratio and temperature
60
40
20
3.0
2.5
2.0
65
20
0
60
55
0
1.5
50
60
250
55
200
50
150
45
1.0
45
100
(c)
100
80
60
40
20
0
65
60
55
50
2.5
2.0
1.5
45
1.0
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J Am Oil Chem Soc (2011) 88:1917–1923
for enzymatic biosynthesis of cetyl 2-ethylhexanoate which
represented higher predicted yield than the others’ in
Fig. 4.
cetyl 2-ethylhexanoate was successfully developed by
RSM and CCRD design.
Obtaining Optimal Synthesis Conditions
Conclusions
The optimal point of synthesis was determined by ridge-
max analysis, which approximates the estimated ridge of
maximum response for increasing radii from the center of
the original design. The ridge-max analysis indicates that
maximum molar conversion was 91.95% at a reaction time
of 2.65 days, a reaction temperature of 56.18 °C, a sub-
strate molar ratio of 2.55:1, and an enzyme amount of
251.39%.
We developed an optimal system for the production of
wax esters by direct esterification of cetyl alcohol with
2-ethylhexanoic acid using immobilized lipase (Nov-
ozymÒ 435) in n-hexane. RSM and 5-level-4-factor
CCRD were employed for the optimization of esterifica-
tion reactions. The ridge-max analysis indicates that
maximum yield was 91.95% at a reaction time of
2.65 days, a reaction temperature of 56.18 °C, a substrate
molar ratio of 2.55:1, and an enzyme amount of 251.39%.
The enzymatic synthesis production of cetyl 2-ethylhex-
anoate can be used as a base oil in cosmetics. In this
study, we found that with branched acid substrates (2-
ethylhexanoic acid) it was more difficult to achieve
esterification via lipase- catalysis. Prolonged reaction time
and large enzyme amounts were required to achieve a
high conversion of cetyl 2-ethylhexanoate.
Model Verification
The adequacy of the predicted model was examined by
additional independent experiments at the suggested opti-
mal synthesis conditions. The predicted yield was 91.95%,
and the actual experimental value was 89.75 1.06%.
Thus, the optimization of lipase-catalyzed synthesis for
Fig. 4 Contour plots of yield of
cetyl 2-ethylhexanoate. The
numbers inside the contour plots
indicate yield under given
reaction conditions
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1923
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