Biocatalytic production of ethyl butyrate from butyric acid with immobilized Candida rugosa lipase on cotton cloth

Article abstract of DOI:10.1016/j.molcatb.2011.05.011

A novel method involving polyethylenimine (PEI) coating and glutaraldehyde cross-linking processes was developed to immobilize Candida rugosa lipase onto cotton cloth. After the systematic investigation, the optimal lipase immobilization was achieved when 0.1 g lipase was loaded on 1.5 g support, which was pretreated with 10 ml of 1.0 mg/ml PEI solution at pH 8.0. Subsequent catalytic analysis of immobilized lipase for ethyl butyrate synthesis was also carried out in the Erlenmeyer flasks. The results indicated that when optimal 0.25 M ethanol and 0.6 M butyric acid were catalyzed by the immobilized lipase at 25 °C, the highest conversion yield of 91.2% and 1.27 mmol h^-1 g^-1 productivity of ethyl butyrate were obtained. Furthermore, a kinetic model of Ping Pong Bi-Bi mode with inhibition of both substrates was proposed and validated by experimental data. To explore the practical potential of immobilized lipase, its operational stability was evaluated and the residual activity was remained about 50% after 12 repeated recycles, with a half-life time of about 300 h for the immobilized lipase. Finally, a recycle batch reactor using immobilized lipase was developed for ethyl butyrate production. The achieved result of 0.85 M final ethyl butyrate concentration, with the conversion of 70.6% and the productivity of 1.45 mmol h^-1 g ^-1, had revealed the promising potential of this immobilized lipase in practical applications.

Full text of DOI:10.1016/j.molcatb.2011.05.011

Journal of Molecular Catalysis B: Enzymatic 72 (2011) 139–144
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biocatalytic production of ethyl butyrate from butyric acid with immobilized
Candida rugosa lipase on cotton cloth
Chengliang Shua, Jin Caia, Lei Huanga,∗, Xiangcheng Zhua,b, Zhinan Xua,∗∗
a Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China
b Hunan Engineering Research Center of Combinatorial Biosynthesis and Natural Product Drug Discovery, Changsha, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel method involving polyethylenimine (PEI) coating and glutaraldehyde cross-linking processes
was developed to immobilize Candida rugosa lipase onto cotton cloth. After the systematic investigation,
the optimal lipase immobilization was achieved when 0.1 g lipase was loaded on 1.5 g support, which was
pretreated with 10 ml of 1.0 mg/ml PEI solution at pH 8.0. Subsequent catalytic analysis of immobilized
lipase for ethyl butyrate synthesis was also carried out in the Erlenmeyer flasks. The results indicated
that when optimal 0.25 M ethanol and 0.6 M butyric acid were catalyzed by the immobilized lipase at
25 ◦C, the highest conversion yield of 91.2% and 1.27 mmol h−1 g−1 productivity of ethyl butyrate were
obtained. Furthermore, a kinetic model of Ping Pong Bi-Bi mode with inhibition of both substrates was
proposed and validated by experimental data. To explore the practical potential of immobilized lipase, its
operational stability was evaluated and the residual activity was remained about 50% after 12 repeated
recycles, with a half-life time of about 300 h for the immobilized lipase. Finally, a recycle batch reactor
using immobilized lipase was developed for ethyl butyrate production. The achieved result of 0.85 M final
ethyl butyrate concentration, with the conversion of 70.6% and the productivity of 1.45 mmol h−1 g−1, had
revealed the promising potential of this immobilized lipase in practical applications.
Received 14 March 2011
Received in revised form 27 May 2011
Accepted 27 May 2011
Available online 6 June 2011
Keywords:
Candida rugosa lipase
Cotton cloth
Polyethylenimine
Substrate inhibition
Ping Pong Bi-Bi
Esterification
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
the point to influence the performance of immobilized enzyme.
Considering about the hydrophobic nature of lipases, it is much
Esters derived from short-chain carboxylic acids such as butyric
acid are a large group of flavor and fragrance compounds, which
are widely used in food, beverage, cosmetic and pharmaceutical
industries [1]. Currently, these esters are mainly synthesized via
environmentally cost chemical routes. However, enzymatic pro-
duction of short-chain esters is gaining an increased attention with
advantages of its mild reaction conditions, high catalytic efficiency
and inherent selectivity [2]. Thus, various lipases have been studied
and applied to catalyze desired ester formation [3–6].
Comparing to free enzymes in the solution, the immobilized
enzyme has some attractive characteristics including the enzyme
reusability and easy accessibility, so this technique has been inten-
sively investigated and utilized in several biocatalysis processes
[7–9]. The success of enzyme immobilization relies on several
important factors such as the amount of enzyme, type of sup-
port as well as the conditions and chemical reagents for the
immobilization. Among them, support is generally considered as
more simple, economic and selective to immobilize lipases onto
hydrophobic supports. However, with increased concentrations of
substrates and higher reaction rates in the support microenviron-
ment, hydrophilic supports such as polyurethane foams were also
competent for lipase immobilization and catalysis of esterification
reaction [2,9]. As an inexpensive and easily available fibrous mate-
rial, cotton cloth had been successfully used in cell and enzyme
immobilization for various fermentations and enzymatic reactions
[10–14]. The usage of cotton cloth in enzyme immobilization could
provide several profits including large immobilization capacity,
simplified configuration, negligible mass transfer resistance, good
operational stability and easy post-processing, which are also valu-
able traits for further industrial application [12]. So far, using cotton
cloth as a viable hydrophilic support for lipase immobilization has
not been reported yet.
Negatively charged cotton cloth contains various chemical
groups including carboxyl, sulfonic, phenolic and hydroxyl. The
direct absorption of enzyme on cotton cloth may cause large loss
of enzyme activity due to the long reaction period for catalysis
and frequent wash process for recycle. Because of the electrostatic
interaction between negatively charged supports, polyethylen-
imine (PEI) has been used as a complementary component for
∗
Corresponding author.
Corresponding author. Tel.: +86 571 87951220; fax: +86 571 87951220.
E-mail addresses: lhuangblue@zju.edu.cn (L. Huang), znxu@zju.edu.cn (Z. Xu).
∗∗
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.05.011

140
C. Shu et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 139–144
cotton cloth
CH2CH2NH
n
Step 1 PEI treated cotton cloth
PEI
Step 2 lipase adsorption by
PEI-coated cotton cloth
CRL
GA
Step 3 cross-linking
O
O
Step 4 washing by distilled water
and drying
immobilized lipase
Fig. 1. Procedure of lipase immobilization on cotton cloth.
the cotton cloth treatment to enhance the efficiency of enzyme
immobilization [15–17]. When PEI-coated cotton cloth was used as
the support, glutaraldehyde (GA) cross-linking was carried out to
finally immobilized enzyme. However, the low density of immobi-
lized enzyme and concomitant substrate inhibition, as well as the
possible deactivation of immobilized enzyme, are the main chal-
lenges need to be solved for the application of this technique in the
biocatalysis of short-chain esters.
In this study, the immobilization of Candida rugosa lipase (CRL)
was carried out, and the immobilization processes were optimized.
Moreover, the performance of immobilized lipase on cotton cloth
was improved via a comprehensive investigation. Several reaction
parameters involved in the synthesis of ethyl butyrate were stud-
ied and a kinetic model was constructed. Finally, the operational
stability of immobilized lipase was examined, and the usage of
immobilized lipase in recycle batch reactor for potential industrial
application was attempted.
for enzyme immobilization, the process was performed in a shaker
with 220 rpm rotation speed at 25 ◦C. In case of recycled batch reac-
tor, the process was performed in a glass column at 25 ◦C, and the
temperature was controlled by water circulation using a peristaltic
pump and a thermostatic water bath. The effects of different factors
on the immobilization of enzyme, including the concentration and
pH of PEI solution, and the amount of cotton cloth, were systemati-
cally investigated. The enzymatic activity and the enzyme recovery
were also determined.
2.3. Esterification reaction
The enzymatic synthesis of ethyl butyrate catalyzed by immo-
bilized lipase (0.6 g) was initially carried out in a 25 ml flask with
10 ml working volume, using cyclohexane as the reaction medium.
The reaction mixture was incubated in a shaker at 220 rpm. The
parameters including the reaction temperature, concentrations of
substrates and molar ratio of substrates (ethanol to butyric acid)
were investigated. Initial reaction rate and conversion yield were
also determined.
2. Materials and methods
2.1. Enzyme and reagents
2.4. Operational stability
C. rugosa lipase type VII was obtained from Sigma, with a quoted
activity of 1449 U/mg of solid. Polyethylenimine (PEI, 80%, ethoxy-
lated, 37 wt% aqueous solution, MW = 50,000) was purchased from
Acros Organics. Glutaraldehyde (GA) as 25% (w/v) aqueous solu-
tion was purchased from Simopharm Chemical Reagent Co. Ltd.,
China. Cotton cloth was obtained locally with specific surface area
of 28–40 m2/g and pore diameter of 2–5 nm. All solutions of PEI,
GA, and enzyme were prepared in distilled water, and the pH was
adjusted using HCl or NaOH solution when necessary.
A batch-wise fashion was adopted to investigate the operational
stability of the immobilized lipase. Each batch was executed for
24 h, and the synthetic activity as well as the conversion yield was
determined. The immobilized lipases were recovered by vacuum
filtration, and washed intensively with cyclohexane. The resulted
enzymes were then mixed with the same volume of fresh reaction
mixture to start a new batch. The experiment was not terminated
until the residual synthetic activity was lower than 50%.
2.5. Analytical methods
2.2. Immobilization of lipase on cotton cloth
Concentrations of ethyl butyrate, ethanol and butyric acid were
determined by the Agilent 6820 gas chromatography, equipped
with a flame ionization detector. Propionic acid was taken as the
inner standard. The conversion yield was calculated according to
the molar ration between the ethyl ester and respective limiting
substrate, either alcohol or acid.
Synthetic activity was measured by ester-synthesis method in
organic media. It was defined as follows: one unit (U) of activity
equals the amount of enzyme catalyzed 1 ␮mol substrate (ethanol
and butyric acid) to form the product (ethyl butyrate) in 1 min
under the assay conditions.
Briefly, the immobilization of lipase was carried out through
three main steps (Fig. 1). 1.5 g cotton cloth was soaked in 10 ml
of 1 mg/ml PEI solution (pH 8.0) for 2 h, and then washed with
distilled water to remove excess PEI. The treated cotton cloth
was wiped with filter paper and soaked into 10 ml of 10 mg/ml
enzyme solution for 1 h. Then the PEI-enzyme-coated cotton cloth
was cross-linked in 10 ml pre-cooled GA solution (1% (w/v), pH
7.0) for 1 h. The resulted cotton cloth with immobilized enzyme
was washed intensively with distilled water, air-dried and con-
served at 4 ◦C until use. When 25 ml Erlenmeyer flask was used

C. Shu et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 139–144
141
Hydrolytic activity of lipase was determined by olive oil emul-
120
100
80
60
40
20
0
70
60
50
40
30
20
10
0
A
sion method [18]. 1 U of lipase hydrolytic activity was defined as
the amount of enzyme that liberated 1 ␮mol of free fatty acid per
minute under the assay conditions. The immobilization yield (%)
was calculated as 1 − Ar/Ai, Ar is the hydrolytic activity of residual
free lipase after immobilization, and Ai is the hydrolytic activity of
initial free lipase.
3. Results and discussion
3.1. Immobilization of C. rugosa lipase on cotton cloth
3.1.1. Effects of the concentration and pH of PEI solution on lipase
immobilization
0
2
4
6
8
C. rugosa lipase is an acidic protein with an isoelectric point of
about 4.4 [19]. The electrostatic attraction and concomitant adsorp-
tion could occur between negatively charged aqueous enzyme
and positively charged PEI-coated cotton cloth. Thus, the inten-
sity of this interaction was directly influenced by the amount
of PEI used and the pH of PEI solution. 10 ml of different con-
centrations (0 mg/ml, 0.1 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 2.0 mg/ml,
4.0 mg/ml and 8.0 mg/ml, all at pH 8) and different pH (6–12, all
with 1.0 mg/ml PEI) of PEI solutions were investigated, respectively,
to treat with a constant 0.6 g cotton cloth. Then 0.1 g lipase powder
was immobilized onto PEI-coated cotton cloth. The immobiliza-
tion efficiency was evaluated with the relative synthetic activity
of loaded enzyme (the maximum value of synthetic activity was
100%) and immobilization yield.
As shown in Fig. 2A, the treatment of PEI could significantly
improve the enzymatic activity and the immobilization yield. When
10 mg PEI was used, the synthetic activity could reach maximum
17.8 U/g immobilized lipase, which was 2.3-fold comparing to
that of the control. Simultaneously, immobilization yield was also
improved with the process of PEI. The maximum immobilization
yield could reach 53.8% when 20 mg PEI was applied. However,
excess addition of PEI over 20 mg could result in the decrease of
both synthetic activity and immobilization yield, which may be due
to the occupation of enzyme binding sites by PEI and the repulsion
betweenPEI. Similarly, withthe presenceof 10 mg PEI, thesynthetic
activity and the immobilization yield both reached their maxi-
mum value at pH 8.0 (Fig. 2B). Considering about the utilization of
immobilized lipase, the optimal condition for immobilization was
determined with 10 mg PEI treatment at pH 8.0.
PEI concentration (mg/ml)
B
120
100
80
60
40
20
0
70
60
50
40
30
20
10
0
5
6
7
8
9
10
11
12
13
pH
Fig. 2. Effect of PEI concentration (A) and pH (B) on lipase immobilization. —(ꢀ)—
Relative synthetic activity; —(᭹)— immobilization yield. The immobilization con-
ditions were: 0.1 g lipase powder, 0.6 g cotton cloth and 10 ml PEI solution. The
same pH value (8.0) was set to test the effect of PEI concentration (0–8.0 mg/ml),
and the same PEI concentration (10 mg/10 ml) was set to test the effect of different
pH value. Esterification conditions: 0.6 g immobilized lipase with 0.3 M ethanol and
0.3 M butyric acid in 10 ml cyclohexane at 25 ◦C.
24 h esterification reaction was monitored (Fig. 4). The concen-
trations of both ethanol and butyric acid were decreased steeply
during the early stage of reaction, because of the adsorption by cot-
ton cloth. The profile of substrate consumption was similar to the
result obtained by using polyurethane foams as the support for CRL
3.1.2. Effect of support amount on lipase immobilization
As the main component, support also contributed to the immo-
bilization procedure. The effects of different amounts of support
(0.5 g, 0.75 g, 1 g, 1.5 g and 2.0 g) were respectively evaluated, with
a constant 0.1 g loading enzyme in 10 ml working volume. The
synthetic activity was enhanced from 9.08 U to 16.7 U when the
amount of support was increased from 0.5 g to 1.5 g (Fig. 3). But
further increase of support amount could lead to the decrease of
synthetic activity, which may be caused by the scattered distri-
bution of enzyme on the support and the resulted less contact
between enzyme and substrate. The immobilization yield gradually
improved with the increase of support amount, and the maximum
yield reached 65% when support amount was 2.0 g (Fig. 3). As a
result, the optimal load ratio between enzyme and support was
1:15.
120
100
80
60
40
20
0
80
60
40
20
0
0.50
0.75 1.00
1.25 1.50
1.75 2.00
3.2. Enzymatic synthesis of ethyl butyrate by immobilized lipase
Cotton cloth (g)
Fig. 3. Effect of support amount on lipase immobilization. —(ꢀ)— Relative synthetic
activity; —(᭹)— immobilization yield. The immobilization conditions: 0.1 g lipase
powder, 10 ml 1.0 mg/ml PEI solution, pH 8.0 and cotton cloth varied from 0.5 g
to 2.0 g. Esterification conditions: 0.6 g immobilized lipase with 0.3 M ethanol and
0.3 M butyric acid in 10 ml cyclohexane at 25 ◦C.
3.2.1. Time-course of esterification reaction catalyzed by
immobilized lipase
With 3.0 g immobilized lipase, 0.5 M ethanol and 0.5 M butyric
acid in a 40 ml reaction system, a comprehensive time course of

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C. Shu et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 139–144
16
14
12
10
8
0.5
0.4
0.3
0.2
0.1
0.0
6
4
2
0
5
10
15
20
25
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Reaction time (h)
Ethanol concentration (M)
Fig. 4. Time course of esterification reaction catalyzed by immobilized lipase at
25 ◦C. —(ꢀ)— Ethanol; —(᭹)— butyric acid; —(ꢁ)— ethyl butyrate. Reaction con-
ditions: 3.0 g immobilized lipase, 0.5 M ethanol and 0.5 M butyric acid in 40 ml
cyclohexane.
Fig. 5. Effect of molar ratio of ethanol to butyric acid on initial reaction rate at
25 ◦C. Butyric acid concentration: —(ꢀ)— 0.1 M; —(᭹)— 0.25 M; —(ꢁ)— 0.4 M; —(ꢀ)—
0.6 M; —(ꢂ)— 0.8 M. The reaction conditions were set in the following: 0.6 g immo-
bilized lipase with appropriate amount of substrates in 30 ml cyclohexane.
immobilization [2]. This so-called positive partition effect behavior
could promote the contact between substrates and enzyme, and
accelerate the reaction. However, excess substrate may result in
the substrate inhibition. The concentration of ethyl butyrate was
increased gradually at a relatively constant rate in the early 8 h, and
then the reaction was slowed down and reached the equilibrium
around 12 h (Fig. 4).
must also be considered. Normally, a reaction should have a suitable
molar ratio of substrates to ensure the best conversion yield. There-
fore, a series of experiments were performed in a binary matrix
mode with five different butyric acid concentrations (0.1 M, 0.25 M,
0.4 M, 0.6 M and 0.8 M) and six different ethanol concentrations
(0.075 M, 0.1 M, 0.25 M, 0.4 M, 0.6 M and 0.8 M). As shown in Fig. 5,
in spite of varied concentration of butyric acid, the highest reac-
tion rates were always achieved when 0.25 M ethanol was initially
presented. The initial reaction rate was steadily enhanced with the
increase of butyric acid concentration up to 0.6 M. However, further
increase of butyric acid could lead to the decrease of initial reaction
rate, which was possibly caused by the substrate inhibition. This
observation was accordant with the one in the previous literatures
[2,21]. So the optimal concentrations of ethanol and butyric acid
were 0.25 M and 0.6 M, respectively, and the educed molar ratio
was 1:2.4.
3.2.2. Effect of reaction temperature on ethyl butyrate synthesis
Different reaction temperatures in the range of 15–42 ◦C were
tested to evaluate its effect on the initial reaction rate and con-
version yield, with 0.6 g immobilized lipase, equimolar of alcohol
and acid (0.1 M) in 30 ml cyclohexane. The initial reaction rate
was improved with the increased temperature up to 37 ◦C, and
the highest reaction rate was 16.7 ␮mol min−1 g−1 (Table 1). Tem-
peratures beyond 37 ◦C resulted in the decrease of initial reaction
rate. Uncoordinatedly, the maximal 87.9% conversion yield was
obtained at 20 ◦C, higher temperature led to the impairment of
conversion yield. Though the higher temperature (less than 37 ◦C)
could enhance the initial reaction rate, the conversion yield was
reduced due to the heat deactivation of enzyme. Considering about
the cost and efficacy, 20–25 ◦C was adopted as the optimal reaction
temperature. Contrast to the reported highest conversion yield of
ethyl butyrate (36% at 37 ◦C) using other support for lipase immo-
3.3. Kinetics of esterification reaction
The investigation of substrates molar ratio had revealed that
both ethanol and butyric acid could act as the inhibitor at the high
concentration. According to previous literatures [21,22], a kinetic
mechanism of Ping Pong Bi-Bi model was proposed with the inhi-
bition of two substrates. A theoretical equation of Ping Pong Bi-Bi
mode with the inhibition of alcohol and acid was presented [23].
v0
=
[acid][alcohol]
[acid][alcohol] + Kmacid[alcohol](1 + [alcohol]/Kialcohol) + Kmalcohol[acid](1 + [acid]/Kiacid
(1)
Vmax
)
In Eq. (1), V0 is the initial reaction rate (␮mol min−1 g−1), Vmax
is the maximal rate, Km is the Michaelis constant of alcohol or acid
(mol/l), Ki is the inhibition constant of alcohol or acid (mol/l). The
data collected from two groups of experiments with 0.4 M or 0.6 M
ethanol, respectively reacted with six different concentrations of
butyric acid (0.075 M, 0.1 M, 0.25 M, 0.4 M, 0.6 M and 0.8 M), were
fitted into the Eq. (1) to determine V0. The kinetic parameters were
calculated using Origin 7.5 software through a nonlinear regression
bilization [6], our result offered a higher conversion yield at lower
temperatures.
3.2.3. Effect of substrate molar ratio on ethyl butyrate synthesis
The equilibrium of esterification could be pushed forward by
the addition of excess alcohol or acid, or by removing products
from the reaction mixture [20], but the effect of substrate inhibition
Table 1
The effects of reaction temperature on initial reaction rate and conversion yield.
Temperature (◦C)
15
20
25
30
37
42
Initial reaction rate (␮mol min−1 g−1
Conversion yield (%)
)
8.8 0.4
82.3 4.1
10.3 0.5
87.9 4.4
14.8 0.7
86.7 4.1
15.5 0.7
85.9 4.3
16.7 0.8
84.5 4.2
15.4 0.7
84.1 4.4

C. Shu et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 139–144
143
15
12
9
6
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Butyric acid concentration (M)
Fig. 6. Initial reaction rate V0 vs. various butyric acid concentrations at constant
ethanol concentrations of 0.4 M and 0.6 M, respectively. Experimental data points:
(ꢀ) 0.4 M ethanol; (᭹) 0.6 M ethanol. Theoretical curve: —— 0.4 M ethanol; —- 0.6 M
ethanol.
analysis. The obtained results were: Vmax = 302.7 ␮mol min−1 g−1
,
Kmalcohol = 0.3 M, Kmacid = 0.09 M, Kialcohol = 0.0041 M, Kiacid = 0.07 M.
The high value of Kiacid/Kialcohol suggested that ethanol is a stronger
inhibitor than butyric acid. As shown in Fig. 4, a dramatic drop
of ethanol concentration steeper than that of butyric acid was
observed during the esterification reaction. The possible reason
may due to the higher hydrophilicity of ethanol comparing to
butyric acid, which makes ethanol easier to be concentrated around
the immobilized lipase on cotton cloth and more competent than
another substrate butyric acid. The comparison of Kmalcohol/Kialcohol
and Kmacid/Kiacid had further confirmed this deduction, which has
also been reported before [24]. As shown in Fig. 6, the experimental
V0 could be approximately fitted with the theoretical model, which
suggested that the Ping Pong Bi-Bi mode is a proper kinetic model
for the synthesis of ethyl butyrate.
Fig. 8. (A) Schematic diagram of the recycle batch reactor for ethyl butyrate syn-
thesis; (B) time-course of esterification reaction using recycle batch reactor. —(ꢀ)—
Ethanol; —(᭹)— butyric acid; —(ꢁ)— ethyl butyrate. Reaction conditions: 36 g immo-
bilized lipase with 1.8 M ethanol and 1.2 M butyric acid in 750 ml cyclohexane,
the reaction temperature was 25 ◦C. A Fed-Batch mode of substrates addition was
adopted.
3.4. Operational stability of immobilized lipase
The operational stability of immobilized lipase was examined
via 12 recycles. As shown in Fig. 7, approximately half of the initial
enzymes maintained their activity even after 12 recycles. There-
fore, the half-life time of the immobilized lipase could reach about
300 h. Comparing to the reported data, the half-life time of immobi-
lized CRL on polyurethane foams was 170.3 h [8], and immobilized
CRL on poly (N-methylolacrylamide) for butyl butyrate synthesis
only had 145 h half-life time [25]. Moreover, the conversion yield
of immobilized lipase was well maintained in the range of 83–92%
among all cycles. In conclusion, the immobilized lipase on cotton
cloth was obviously more endurable to the recycle operation, with-
out significant loss of its conversion efficiency.
120
100
100
80
60
40
80
60
3.5. Ethyl butyrate synthesis in a recycle batch reactor system
40
To explore the industrial potential and feasible application of
immobilize lipase for ethyl butyrate synthesis, a recycle batch
reactor was designed (Fig. 8A). Approximately 36 g immobilized
lipase was packed into the column to catalyze the synthesis of
ethyl butyrate, with the presence of 1.2 M butyric acid and 1.8 M
ethanol in cyclohexane. The reaction mixture was supplied follow-
ing a Fed-Batch mode to eliminate the substrates inhibition. As
shown in Fig. 8B, 0.85 M ethyl butyrate was produced after 97 h
reaction, with the conversion yield of 70.6% and the productivity
of 1.45 mmol h−1 g−1. Comparing to other system, the much higher
20
0
20
0
0
2
4
6
8
10
12
Number of cycles
Fig. 7. Operational stability of immobilized lipase on cotton cloth at 25 ◦C. —(ꢀ)—
Residual activity; —(᭹)— conversion yield. Reaction conditions: 0.6 g immobilized
lipase with 0.3 M ethanol and 0.3 M butyric acid in 10 ml cyclohexane.

144
C. Shu et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 139–144
concentration of final product had indicated a promising industrial
application of this recycle batch reactor.
(Grant No. 20736008) and the Natural Science Foundation of Zhe-
jiang Province (Grant No. R4090041), the People’s Republic of
China.
4. Conclusions
References
A practical immobilization method was developed to load lipase
onto PEI-coated cotton cloth. Several factors including the amount
of PEI, the pH of PEI solution and the amount of support were inves-
tigated to improve the immobilization efficiency. The obtained
immobilized lipase was then applied to the synthesis of ethyl
butyrate, for the evaluation of its enzymatic performance. The
molar ratio of substrates and the reaction temperature were opti-
mized to maximize the conversion yield of immobilized lipase.
A kinetic mechanism of Ping Pong Bi-Bi model with inhibition of
both substrates was proposed and validated by experimental data.
Moreover, with the validated good operational stability of immo-
bilized lipase, a recycle batch reactor was established to make an
attempt on the possible industrial application of immobilized lipase
for ethyl butyrate production. The achieved result of 0.85 M final
ethyl butyrate concentration, with the conversion of 70.6% and the
productivity of 1.45 mmol h−1 g−1, had revealed the great poten-
tial of this immobilized lipase in the practical applications of ester
production. Moreover, the bio-production of butyric acid via the
fermentation of Clostridium tyrobutyricum was greatly hampered
by the subsequent separation of product from fermentation broth,
due to the high boiling point (163.5 ◦C) of butyric acid. Consider-
ing about the lower boiling point of ethyl butyrate (121.3 ◦C), the
post-processing of C. tyrobutyricum fermentation broth via immo-
bilized lipase could be proposed to not only facilitate the isolation of
butyric acid, but also directly generate the useful ethyl butyrate for
industrial applications. Thus, the system established in this paper
has promising capability to develop the bio-production of butyric
acid and its ester derivatives.
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Acknowledgements
This work was financially supported by the Ministry of Sci-
ence and Technology of China (Grant Nos. 2009AA02Z206 and
2007CB707805), the National Natural Science Foundation of China