Enhancement of activity and stability of lipase by microemulsion-based organogels (MBGs) immobilization and application for synthesis of arylethyl acetate

Article abstract of DOI:10.1016/j.molcatb.2012.02.005

Lipase from Candida rugosa (CRL) was immobilized in microemulsion-based organogels (MBGs) and subsequently applied in large scale synthesis of arylethyl acetate in organic solvents as a more stable and efficient catalyst. Various reaction parameters (solvent, temperature, substrate concentration) were investigated for enhancement of ester production. Thermal and operational stabilities were improved compared with free CRL showing its potential for continuous applications. They were more stable at 50-60 °C and showed good recovery activity, which retained 70% of their initial activity after 16 recycles in organic media and remained constant at that level thereafter. Moreover, the immobilized lipase can maintain high catalytic activity in a variety of organic solvents, while free lipase was easily inactivated in polar solvent. A series of alcohols with different substitution groups were successfully applied in CRL MBGs-catalyzed transesterification, affording higher conversions than those with the free enzyme.

Full text of DOI:10.1016/j.molcatb.2012.02.005

Journal of Molecular Catalysis B: Enzymatic 78 (2012) 65–71
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enhancement of activity and stability of lipase by microemulsion-based
organogels (MBGs) immobilization and application for synthesis of arylethyl
acetate
Wei-Wei Zhang, Na Wang∗, Yu-Jie Zhou, Ting He, Xiao-Qi Yu∗
Key Laboratory of Green Chemistry & Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipase from Candida rugosa (CRL) was immobilized in microemulsion-based organogels (MBGs) and
subsequently applied in large scale synthesis of arylethyl acetate in organic solvents as a more stable
and efficient catalyst. Various reaction parameters (solvent, temperature, substrate concentration) were
investigated for enhancement of ester production. Thermal and operational stabilities were improved
compared with free CRL showing its potential for continuous applications. They were more stable at
50–60 ◦C and showed good recovery activity, which retained 70% of their initial activity after 16 recycles
in organic media and remained constant at that level thereafter. Moreover, the immobilized lipase can
maintain high catalytic activity in a variety of organic solvents, while free lipase was easily inactivated
in polar solvent. A series of alcohols with different substitution groups were successfully applied in CRL
MBGs-catalyzed transesterification, affording higher conversions than those with the free enzyme.
© 2012 Elsevier B.V. All rights reserved.
Received 22 October 2011
Received in revised form 20 January 2012
Accepted 13 February 2012
Available online 22 February 2012
Keywords:
Lipase
Immobilization
Microemulsion
Transesterification
1. Introduction
approaches to resolve these problems, one of the most effective
methods is immobilization of enzymes within an aqueous microen-
The potential of biocatalysis has been increasingly recognized
as an efficient and green tool for modern organic synthesis due to
the high selectivity and specificity under mild reaction conditions.
Thus, the application of enzymes represents a remarkable opportu-
nity for the development of industrial chemical and pharmaceutical
processes [1–7]. Among these enzymes, lipases undoubtedly play
an important role and are widely used in synthetic organic chem-
istry due to their high activity, wide sources and broad range of
substrates [8,9]. In particular, the discovery of enzymatic activity
in organic solvents represented a major achievement, presenting
biocatalysis as a practical tool for the preparation of organic com-
pounds [10–13]. Some problems associated with the poor solubility
of reactants, side reactions such as hydrolysis or decomposition and
difficult product recovery in aqueous medium have been usually
overcome.
vironment in the organic solvents [15,16].
Microemulsions formed by amphiphilic molecules have been
widely reviewed as effective media for enzyme reactions requiring
hydrophobic solvents [17]. Interestingly, the microemulsions have
been developed as novel immobilization supports for enzymes
by adding some natural resource polymers such as gelatin
[18]. Gelled microemulsions, i.e. microemulsion-based organogels
(MBGs), have become a subject of interest as catalysts for lipase-
catalyzed lipid transformations [19,20]. Recently, Zoumpanioti
et al. reviewed several MBGs based on different matrices for vari-
ous enzyme immobilizations and summarized the parameters that
affect the use of MBGs as media for organic reactions catalyzed by
lipases [21]. The MBGs prepared by biopolymers and microemul-
sions were used as matrices for enzyme immobilization in order to
achieve enzymatic catalysis in non conventional medium as they
appeared to be rigid and stable for a long time, even within the
reaction solution [22]. Raghavendra et al. reported Candida rugosa
lipase immobilized in microemulsion based organogels (MBGs) as
an efficient biocatalytic system for esterification reactions of ethyl
valerate in organic solvents [23].
However, enzymes often display drastically lower activity in
organic solvents than in water. It is believed that the water layer
on the molecular surface of enzymes determines their activity
in organic media, and there are three major causes of the low
activity—unfavorable substrate desolvation, suboptimal pH, and
reduced conformational mobility [5,14]. Among several known
Arylethyl acetate is a kind of important ester used for the prepa-
ration of rose, jasmine and hyacinth fragrance [24–26]. So it is an
essential additive in producing soap, detergents, cosmetics, sprays,
deodorants and so on. It is also used as food flavors and mainly
for the preparation of fruit, cream, caramel, honey and vanilla
aromas. However, the conventional synthesis of arylethyl acetate
∗
Corresponding author. Tel.: +86 28 85415886; fax: +86 28 85415886.
E-mail addresses: wnchem@scu.edu.cn (N. Wang), xqyu@scu.edu.cn (X.-Q. Yu).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2012.02.005

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W.-W. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 65–71
using sulfuric acid or phosphoric acid [27–29] as catalyst has led to
numerous side reactions with low product quality, and even serious
equipment corrosion. Therefore, developing a green and efficient
biocatalytic method has been an urgent demand.
petriplates and kept for air drying overnight. Next day the dried gel
was cut into small pieces and used for transesterification reaction.
2.3. Enzymatic reaction condition
In the present study, we demonstrate Candida rugosa lipase
immobilized in gelatin MBGs as a more effective and stable bio-
catalyst for the transesterification of arylethanol and vinyl acetate.
Various parameters such as surfactants, reaction medium and tem-
perature were optimized. It is noted that the CRL MBGs showed
higher thermostability and reusability than free CRL, which is
important for its application in the chemical industry. Furthermore,
we have also demonstrated that lipase retained high activity in
gram scale synthesis of arylethyl acetate.
In addition to exploring the effect of solvents, other experi-
ments were carried out in solvent-free systems. MBGs containing
lipases were weighed and added to 2 mL of vinyl acetate containing
10 mg/mL 2-phenylehthanol unless otherwise noted. The reaction
mixture was incubated with shaking at the desired temperature
and 150 rpm in a temperature-controlled shaker. At regular time
intervals, samples were withdrawn from the reaction medium
and analyzed by HPLC. In order to determine the substrate scope,
a series of alcohols with different substitution groups were also
investigated using immobilized and free enzyme. The transesteri-
fication reaction of large scale with 5 g and 10 g 2-phenylehthanol
were carried out as described in Section 3.7. All the experiments
were repeated at least three times.
2. Materials and methods
2.1. Materials
Sodium bis-2-(ethylhexyl) sulfosuccinate (AOT) were obtained
from Acros (USA). Candida rugosa lipase (triacylglycerol acyl hydro-
lase, E.C. 3.1.1.3, type VII, 807 U/mg) and Gelatin (from porcine skin,
type II) were purchased from Sigma (USA) and both used without
further purification. All organic solvents and other chemicals used
were of analytical reagent grade. Double distilled water was used
throughout the experiments.
2.4. Effect of different lipase concentration of MBGs
The optimum lipase concentration of encapsulated lipase was
determined as the activity under a range of CRL concentration
(5 mg/mL, 10 mg/mL, 15 mg/mL and 20 mg/mL).
2.5. Effect of solvents and temperature on transesterification
Free and encapsulated lipase preparations were used for trans-
esterification in various organic solvents. Isopropyl ether, dioxane,
toluene, THF, hexane, tert-butyl alcohol, acetone, acetonitrile were
selected as reaction medium.
Effect of temperature was studied by carrying out the reac-
tion at eleven different temperatures: 10, 15, 20, 25, 30, 37, 40,
45, 50, 55 and 60 ◦C. The reaction system consisted of MBGs of
AOT/buffered lipase/isooctane in 2 mL vinyl acetate containing
10 mg/mL 2-phenylehthanol. A control was maintained at each
temperature with free lipase as the catalyst. In the test of studying
the time course of CRL and MBGs at 37 ◦C and 50 ◦C, the reaction
was carried out in 50 mL vinyl acetate.
2.2. Preparation of different surfactants based MBGs
2.2.1. Preparation of surfactant coated lipase
Surfactant coated lipase was prepared according to the method
of Goto et al. [30]. For the preparation of lipase solution, 150 mg
CRL was added in 10 mL of 0.1 M phosphate buffer (pH 7.2) and
shaken for 60 min under 4 ◦C. Surfactant coated lipases were pre-
pared using anionic (AOT), cationic (CTAB) and nonionic (Triton
X-100) surfactants at 1 mM concentration, respectively. Then in the
presence of surfactant, lipase solution was incubated overnight at
4 ◦C. After 24 h, the lipase solution was added to the following pre-
pared reverse micellar solutions using corresponding surfactants,
respectively.
2.6. Effect of 2-phenylehthanol concentration on
transesterification
2.2.2. Preparation of microemulsions using different surfactants
Thermodynamically stable reverse micellar solution was pre-
pared by mixing each component in a suitable ratio according
to the procedure of Dandavate and Madamwar [31]. Anionic,
cationic and non-ionic reverse micellar solutions were pre-
pared using AOT (AOT/buffer/isooctane, Wo = 60 [32]), CTAB
(CTAB/buffer/isooctane/1-hexanol, Wo = 20, Po = 7.8) and Triton X-
100 (Triton X-100/buffer/isooctane/1-hexanol, Wo = 20, Po = 4.2
[33]), respectively which were detailed explained by Raghavendra
et al. [23]. Lipases containing microemulsions were prepared by the
addition of the above prepared aqueous lipase solution (containing
150 mg solid lipase) to different reverse micellar solutions (surfac-
tant molarity 0.1 M) of appropriate water content in the isooctane.
The buffer consisted of 100 mM NaH2PO4–Na2HPO4 solution (pH
7.2).
For evaluation of the effect of 2-phenylehthanol concentration
on transesterification, the concentration of vinyl acetate was main-
tained constant while the concentration of 2-phenylehthanol was
varied from 1 mg/mL to 20 mg/mL. The reaction system consisted
of MBGs of AOT/buffered lipase/isooctane in 50 mL vinyl acetate
containing different concentrations of 2-phenylehthanol. A control
was maintained at each temperature with free lipase as the catalyst.
2.7. Reusability of the MBGs
For reusability studies, the transesterification reaction was car-
ried out as described above for phenylethyl acetate, and upon
completion of one cycle, the immobilized enzyme was recovered
by filtration. Then the MBGs were washed 2–3 times with solvent
for complete removal of substrates and product. Then the solvent
was removed with N2 and reintroduced into a fresh medium. This
procedure was repeated for several cycles.
2.2.3. Preparation of MBGs
The microemulsion-based organogels were prepared by intro-
ducing appropriate amounts of microemulsion containing the
lipases, to a second solution of gelatin in buffer at 55 ◦C. Gelatin
obtained from porcine skin was dissolved in 0.1 M phosphate
buffer (pH 7.2), autoclaved and cooled down to 55 ◦C. The result-
ing mixture was vigorously shaken and stirred until homogeneous
and cooled to 25 ◦C. Then the mixture was poured into plastic
2.8. Analytical procedure
The quantitative analysis of samples was carried out by HPLC on
a reverse phase column (Welchrom-C18, 5 ␮m, 4.6 mm × 150 mm,
Welchrom) using a Shimadzu LC-2010A HT apparatus equipped

W.-W. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 65–71
67
with a UV detector at 254 nm. Methanol containing 30% (v/v) water
was used as the mobile phase and at a split flow rate of 0.8 mL/min.
Ester identification and quantification were done by comparing the
retention time and peak area of the sample with standard. Pure
phenylethyl acetate was used as external standard.
1H NMR and 13C NMR spectra were obtained on a Bruker DMX
400. Spectra were run in CDCl3 and referenced to an internal TMS
standard.
Scheme 1. Transesterification of 2-phenylethanol and vinyl acetate.
In microemulsions and MBGs systems, electrostatic interac-
tion between amphiphilic molecules and enzyme molecules at the
interface could play an important role on the enzyme activity [21].
Surfactants behave as the interface between the organic phase and
the enzyme harboring buffer providing first level of protection to
the enzyme entrapped within the micelle [23]. So the influence
of the different kinds of surfactants used for gelatin MBGs prepa-
ration was investigated by immobilizing lipase from C. rugosa in
CTAB (cationic), AOT (anionic) and Triton X-100 (nonionic) based
microemulsion organogels, respectively. All the three surfactants
formed thermostable reverse micellar solutions with the solvents.
From Fig. 1, AOT based MBGs gave the highest yield with 98.4%
transesterification after 24 h in comparison to CTAB or Triton X-
100 based MBGs. CTAB based MBGs performed the lowest activity,
which may be due to the strong interaction between the cationic
head group in the surfactant molecule and the negatively charged
lipase. The interaction might induce change in three-dimensional
structure of lipase which may result in reduced activity [34]. Com-
pared with cationic surfactants, moderate interactions between
lipase and nonionic surfactant are responsible for better catalytic
activity than cationic surfactants. But different observations were
made by Dandavate and Madamwar [31]. In their study, it was
found that lipase immobilized in Triton X-100 based organogels
exhibited comparatively lower esterification activity than lipase
immobilized in CTAB-based organogels. They speculated that it
might be owing to the poor stability of Triton X-100 organogels.
However in our study we obtained stable Triton X-100 organogels
with different Wo and Po value which may perform better transes-
terification activity. Thus AOT based MBGs were further exploited
at various conditions.
2.9. Characterization of products
Phenylethyl acetate (1): 1H NMR (400 MHz, CDCl3, ı, ppm):
7.32–7.21 (m, 5H), 4.28 (t, 2H, J = 8.0 Hz), 2.94 (t, 2H, J = 6.0 Hz), 2.03
(s, 3H); 13C NMR (100 MHz, CDCl3, ı, ppm): 170.96, 137.87, 128.92,
128.54, 126.60, 64.95, 35.13, 20.93. ESI-MS: 165.1 [M+H]+.
2-Phenylpropyl acetate (2): 1H NMR (400 MHz, CDCl3, ı, ppm):
7.33–7.21 (m, 5H), 4.21–4.10 (m, 2H), 3.09 (q, 1H, J = 8.0 Hz), 2.01
(s, 3H), 1.30 (d, 3H, J = 8.0 Hz); 13C NMR (100 MHz, CDCl3, ı, ppm):
170.96, 143.22, 128.52, 127.30, 126.71, 69.39, 38.95, 20.86, 18.10.
ESI-MS: 179.1 [M+H]+.
Benzyl acetate (3): 1H NMR (400 MHz, CDCl3, ı, ppm): 7.37–7.34
(m, 5H), 5.11 (s, 2H), 2.10 (s, 3H); 13C NMR (100 MHz, CDCl3, ı,
ppm): 170.94, 135.96, 128.59, 128.29, 66.34, 21.02. ESI-MS: 151.1
[M+H]+.
4-Bromobenzyl acetate (4): 1H NMR (400 MHz, CDCl3, ı, ppm):
7.50–7.22 (m, 4H), 5.05 (s, 2H), 2.10 (s, 3H); 13C NMR (100 MHz,
CDCl3, ı, ppm): 170.65, 135.02, 131.68, 129.91, 122.23, 65.43, 20.92.
ESI-MS: 251.0 [M+Na]+.
3-Methylbenzyl acetate (5): 1H NMR (400 MHz, CDCl3, ı, ppm):
7.27–7.13 (m, 4H), 5.07 (s, 2H), 2.36 (s, 3H), 2.10 (s, 3H); 13C NMR
(100 MHz, CDCl3, ı, ppm): 171.02, 138.28, 135.88, 129.06, 125.40,
66.43, 21.37, 21.01. ESI-MS: 187.1 [M+Na]+.
1-Phenylethyl acetate (6): 1H NMR (400 MHz, CDCl3, ı, ppm):
7.36–7.29 (m, 5H), 5.88 (q, 1H, J = 8.0 Hz), 2.07 (s, 3H), 1.53 (d,
3H, J = 4.0 Hz); 13C NMR (100 MHz, CDCl3, ı, ppm): 170.31, 141.73,
128.53, 127.89, 126.12, 72.33, 22.23, 21.34. ESI-MS: 165.1 [M+H]+.
4-Nitrobenzyl acetate (7): 1H NMR (400 MHz, CDCl3, ı, ppm):
8.23 (d, 2H, J = 8.0 Hz), 7.52 (d, 2H, J = 8.0 Hz), 5.21 (s, 2H), 2.16 (s,
3H); 13C NMR (100 MHz, CDCl3, ı, ppm): 170.50, 147.63, 143.30,
128.35, 123.73, 64.73, 20.78. ESI-MS: 218.0 [M+Na]+.
3.2. Effect of different lipase concentration of MBGs
Theeffectoftheconcentrationof enzymeusedduringtheimmo-
bilization step was studied and the result was shown in Fig. 2. We
investigated different lipase concentration of MBGs on transesteri-
fication. From Fig. 2, it was observed that the transesterification rate
decreased markedly as the amount of lipase used in immobiliza-
tion processes increased. Excessive lipase concentration resulted in
Cyclohexyl acetate (8): 1H NMR (400 MHz, CDCl3, ı, ppm):
4.74–4.72 (m, 1H), 2.03 (s, 3H), 1.86–1.67 (m, 4H), 1.42–1.26 (m,
6H); 13C NMR (100 MHz, CDCl3, ı, ppm): 170.46, 72.58, 31.60, 25.33,
23.75, 21.33. ESI-MS: 143.1 [M+H]+.
Octyl acetate (9): 1H NMR (400 MHz, CDCl3, ı, ppm): 4.05 (t,
2H, J = 6.0 Hz), 2.05 (s, 3H), 1.64–1.59 (m, 2H), 1.36–1.26 (m, 10H),
0.88 (t, 3H, J = 6.0 Hz); 13C NMR (100 MHz, CDCl3, ı, ppm): 171.11,
64.58, 31.74, 29.18, 29.14, 28.57, 22.59, 20.90, 14.00. ESI-MS: 173.2
[M+H]+.
Decyl acetate (10): 1H NMR (400 MHz, CDCl3, ı, ppm): 4.05 (t,
2H, J = 8.0 Hz), 2.05 (s, 3H), 1.83–1.58 (m, 2H), 1.31–1.27 (m, 14H),
0.88 (t, 3H, J = 8.0 Hz); 13C NMR (100 MHz, CDCl3, ı, ppm): 171.09,
64.58, 31.85, 29.49, 29.26, 29.22, 28.58, 25.88, 22.63, 20.89, 14.02.
ESI-MS: 201.2 [M+H]+.
3. Results and discussion
3.1. Synthesis of arylethyl acetate catalyzed by CRL immobilized
in MBGs using different surfactant based reverse micelles
The CRL immobilized in MBGs using different surfactants were
applied and the activities of these biocatalysts were evaluated
in the transesterification of 2-phenylethanol with vinyl acetate
(Scheme 1).
Fig. 1. Effect of different surfactants based reverse micelles. (a) Transesterification
was carried out at 37 ◦C and 150 rpm for 24 h in vinyl acetate. 2-Phenylethanol
concentration was 10 mg/mL.

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W.-W. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 65–71
Fig. 2. Effect of different lipase concentration of MBGs. (a) Transesterification was
carried out at 37 ◦C and 150 rpm for 24 h in vinyl acetate. 2-Phenylethanol concen-
tration was 10 mg/mL.
Fig. 3. Effect of solvents on transesterification. (a) Transesterification was carried
out at 37 ◦C and 150 rpm for 24 h. 2-Phenylethanol concentration was 10 mg/mL.
(Slash) CRL; (filled) CRL immobilized in MBGs.
considerable damage to the reverse micelle solution and the
balance among the lipase molecules leading to aggregation or
precipitation. High lipase concentration promoted the lipase
molecules to form strong bimolecular aggregates which get easily
disrupted into monomolecular forms in the presence of surfactant.
Surfactants are known to increase the activity of lipases and are
proposed to act by causing disaggregation of bimolecular forms of
enzyme, promoting interfacial adsorption and shifting the lipase
conformational equilibrium towards the open form [35]. So in the
same reverse micelle solution with lower lipase concentration, sur-
factants can contact with the enzyme sufficiently, thus improving
the lipase activity remarkably. This may be the major reason for
higher activity of MBGs with lower lipase concentration in trans-
esterification.
appropriate solvents for gelatin gels, which is in agreement with
our study. Moreover, the same reaction was also carried in solvent-
free system with good performance. In this case, 2-phenylethanol
was solubilized in excess of vinyl acetate. Lipase-containing MBGs
based on gelatin did not dissolve any of their components signifi-
cantly in vinyl acetate. It should be noted that the MBGs retained
their integrity in all solvents used, even in acetonitrile compared
with the control. This indicates that the stability of the immobilized
lipase has been greatly improved. Thus vinyl acetate was chosen as
reactant and solvent in the further research.
3.4. Effect of temperature on transesterification
The effect of temperature on free and encapsulated lipases was
given in Fig. 4. As seen in Fig. 4, in the range of 10–60 ◦C, the immobi-
lized enzyme can maintain high activity, while the free lipase used
as control showed extremely low ester production at higher tem-
peratures. Optimum temperature interval of free lipase was found
to be 20–40 ◦C and when the temperature was higher than 40 ◦C its
activity declined sharply while immobilized lipase remained sta-
ble. It was conveyed that effective protection to lipase offered by
immobilization process against thermal denaturation.
3.3. Effect of solvents on transesterification
The reaction medium plays a significant role in maintaining the
catalytic activity and stability of an enzyme [36–39]. It has been
reported that suspension of enzymes in organic solvents results in
conformational changes and thereby the specificity of substrates
[40]. The catalytic activity of lipase was remarkably influenced by
different media (Fig. 3). The yields of reaction essentially followed
the pattern of the logarithm of the partition coefficient (log P) that
is used to characterize the solvents in terms of hydrophobicity. In
general, the catalytic activity of the CRL immobilized in MBGs was
improved in most solvents, especially in more polar solvent. As
seen in Fig. 3, in all the solvents used, immobilized CRL can main-
tain more than 40% yields, while there was virtually no product
observed in dioxane, THF, acetone and acetonitrile for the trans-
esterification catalyzed by free CRL. It has been hypothesized that
polar solvents strip away the essential water bound to the enzyme
by participating in non-covalent solvent protein interaction, result-
ing in their active conformational distortion.
However, immobilized lipase showed retention of most activ-
ity in all solvents. It was proposed that immobilization of lipase in
MBGs can retain activity and enhance their stability in polar solvent
due to the enzyme entrapped in the water core of the microemul-
sion based on gel matrix and did not contact with solvent directly.
On the contrary, non-polar solvents such as hexane, toluene, iso-
propyl ether, showed higher performances. This can be attributed
to the extreme hydrophobic nature of the solvent stabilizing the
hydrated enzyme structure. In a more recent work by Dave and
Madamwar [33], n-hexane and isooctane were reported as the most
Fig. 4. Effect of temperature on transesterification. (a) Transesterification was
carried out at various temperatures and 150 rpm for 24 h in 2 mL vinyl acetate.
2-Phenylethanol concentration was 10 mg/mL. (ꢀ) CRL; (᭹) CRL immobilized in
MBGs.

W.-W. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 65–71
69
Fig. 5. Ester synthesis using CRL (ꢀ, ꢀ) and MBGs (ꢁ, ᭹) at 37 ◦C (hollow sym-
bols) and 50 ◦C (solid symbols). aTransesterification was carried out at 37 ◦C or 50 ◦
C
Fig. 6. Effect of 2-phenylethanol concentration on transesterification. (a) Transes-
terification was carried out at 50 ◦C and 150 rpm for 48 h in 50 mL vinyl acetate.
(ꢁ) 1 mg/mL; (᭹) 2 mg/mL; (ꢀ) 5 mg/mL; (ꢂ) 10 mg/mL; (ꢀ) 20 mg/mL; (dotted line)
CRL; (solid line) MBGs.
and 150 rpm for 96 h in 50 mL vinyl acetate. 2-Phenylethanol concentration was
2 mg/mL.
Fig. 5 represents the lipase-catalyzed transesterification of 2-
phenylethanol with vinyl acetate at 37 ◦C (hollow symbols) and
50 ◦C (solid symbols) as a function of time. The concentrations of
both substrates and products were analyzed by HPLC at different
time intervals. Under 37 ◦C, the activity of free lipase was higher
than MBGs at the beginning, which is due to substrate diffusion in
the immobilized enzyme limited by mass transport phenomena.
As the reaction progresses, substrate around immobilized lipase
achieved the balance of diffusion. Mass transport phenomena no
longer affect the reactivity of the immobilized lipase. At higher
temperature, most of the free enzyme was inactivated, while the
immobilized enzyme still maintained a good activity. At 50 ◦C, the
reaction rate of CRL MBGs was significantly improved when com-
pared to that at 37 ◦C. This fact suggests that at higher temperatures,
the conversion rate enhanced strongly with increase in tempera-
ture which indicates that the conversion rate is controlled by the
reaction steps, rather than the mass transport phenomena of the
diffusion of the substrates in the interior of the catalyst. However, at
lower temperatures, the reaction rate is not limited by the reaction
steps but by mass transport phenomena which can be attributed to
the slow increase in ester production with time increasing [41].
reached the balance, while the reaction rate of free lipase reduced
gradually.
For the immobilized enzyme, the conversion increased with
substrate concentration decreasing, the best substrate concentra-
tion was found to be 1 mg/mL as well as free lipases. But for the free
enzyme, the influence of substrate concentration on the conversion
had no obvious patterns.
3.6. Reusability of the MBGs
For any applications based on immobilized enzymes, the fea-
sibility of regeneration of the enzyme activity and consequent
reuse of the biocatalyst are beneficial for its industrial productions
[42]. Reusability for the immobilized enzyme is very important in
economics, and an improved stability can make the immobilized
enzyme more advantageous than its free form. In order to deter-
mine the efficiency of immobilization, the MBGs were subjected
to reusability examination, as it offers the benefits of the reuse of
enzyme and easy separation of products from enzyme as well as the
potential to run a continuous process. To investigate the reusabil-
ity, the enzymes immobilized were washed 2–3 times with vinyl
acetate then dried with N2 and added into a fresh reaction medium
at 50 ◦C. Fig. 7 shows that the activity of CRL MBGs was not sig-
nificantly affected up to 8 runs, after which their activity started
declining and maintained 70% of their original activities after 16
runs, respectively. These results indicated better immobilization
efficiency and reaction systems depicted by the high reaction rates
even after 15–16 runs as compared to others. No accumulation of
excess water formed as a byproduct of transesterification reaction
may be account for this result.
3.5. Effect of 2-phenylehthanol concentration on
transesterification
As substrate concentration is a very significant parameter in any
enzymecatalyzedreaction, it is importantto determinetheireffects
on the enzyme and reaction kinetics. Fig. 6 shows the effect of 2-
phenylethanol concentration on transesterification.
To determine the optimum concentration of 2-phenylethanol,
vinyl acetate was used as solvent. As can be seen in Fig. 6, it was
concluded that the productivities of immobilized enzymes were
much higher than the free enzymes at all substrate concentrations.
Optimum 2-phenylethanol concentration was found to be 1 mg/mL,
showing 100% product formation after 24 h whereas the reactions
proceeded till 48 h at other concentrations. Also, free lipase used as
control showed extremely low production at the same conditions.
Because of the substrate diffusion limitation, the initial reaction
rates of immobilized enzymes were lower than that of free enzymes
in a variety of substrate concentrations (data not shown). However,
for its superior stability against organic solvent and high temper-
ature, the conversion of the immobilized enzyme had increased
significantly after the substrate diffusion in the immobilized lipase
3.7. Substrate scope and large-scale synthesis of arylethyl acetate
To explore the substrate scope, a series of alcohol with different
substitution groups reacted smoothly with vinyl acetate under CRL
MBGs-catalyzed transesterification. The results of this screening
were summarized in Table 1. Among the substrates tested, primary
alcohols with aromatic groups (Table 1, Entries 1–5, 7) showed dra-
matically high yield. Apparently, aromatic group is crucial for the
transesterification. On the other hand, increasing the steric demand
in the 2-position by changing from hydrogen to methyl (Entry 2)
affected the reaction only marginally and the effect of variations at
the aryl moiety in the 4-position of the aryl (Table 1, Entries 4, 7)

70
W.-W. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 65–71
Table 1
CRL MBGs catalyzed transesterification of alcohols.a
Entry
Substrate
Yield (%)
99.4
Entry
6
Substrate
Yield (%)
48.0
OH
OH
OH
1
OH
O2N
2
3
96.1
99.8
7
8
79.7
43.8
OH
OH
OH
OH
Br
3
4
97.9
9
56.0
52.5
OH
CH3
OH
4
5
98.9
10
a
Reactions performed with 0.2 mmol alcohols, 25 mL vinyl acetate and 1 mg/mL CRL MBGs at 50 ◦C for 48 h.
under optimum conditions. Several parameters were optimized to
achieve optimal biocatalytic operation. We have clearly demon-
strated that immobilization of C. rugosa lipase using AOT based
MBGs remarkably improved its stability for organic solvent and
high temperature. This system permitted the reuse of enzyme up to
16 cycles of reaction without any significant loss in activity. The low
catalyst loading and mild reaction conditions demonstrated higher
efficiency of this transesterification, which seems very promising
for lipase-catalyzed, large-scale production of arylethyl acetate fra-
grance.
Acknowledgments
This work was financially supported by the National Natu-
ral Science Foundation of China (No. 21001077 and 21021001),
the Program for Changjiang Scholars in China. We also thank the
Sichuan University Analytical & Testing Center for NMR analysis.
Fig. 7. Reusability of the MBGs. aTransesterification was carried out at 50 ◦
and 150 rpm for 24 h in vinyl acetate and the MBGs was recycled 16 times. 2-
Phenylethanol concentration was 10 mg/mL.
C
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