Enhancement of transesterification-catalyzing capability of bio-imprinted tannase in organic solvents by cryogenic protection and immobilization

Article abstract of DOI:10.1016/j.molcatb.2013.04.007

Improvement of transesterification-catalyzing capability of bio-imprinted tannase is a crucial question of whether to be efficiently utilized in organic media. As for biotransformation of tannic acid to propyl gallate, bio-imprinting technique can dramatically enhance the transesterification-catalyzing capability of tannase. In this work, both cryogenic protection and immobilization were utilized to further improve its apparent catalytic capability in organic media. The results show that Triton-X-100, mannose, and magnesium ion all have a positive effect on cryogenic protection of the tannase. Particularly, combinational application of the three cryoprotectants increases its catalytic performance by 2.7-fold factor. Also, immobilization further elevates its catalytic capability by 2.1 folds. Noteworthily, the coupling application of immobilization and cryo-protection can cause the conversion rate of substrate of the bio-imprinted tannase to increase to a promising 70%. Consequently, it will be helpful to fully utilize tannase in organic phase.

Full text of DOI:10.1016/j.molcatb.2013.04.007

Journal of Molecular Catalysis B: Enzymatic 94 (2013) 1–6
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enhancement of transesterification-catalyzing capability of bio-imprinted
tannase in organic solvents by cryogenic protection and immobilization
Guangjun Niea,b,c, Zhen Chena,1, Zhiming Zheng , Wei Jin , Guohong Gong , Li Wang , Wenjin Yue
a,∗
a
a
a
b,∗
a
Key Lab of Ion Beam Bioengineering, Chinese Academy of Science, 230031, Hefei, China
College of Biochemical Engineering, Anhui Polytechnic University, 241000, Wuhu, China
School of Life Science, University of Science and Technology of China, 230022, Hefei, China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Improvement of transesterification-catalyzing capability of bio-imprinted tannase is a crucial question
of whether to be efficiently utilized in organic media. As for biotransformation of tannic acid to propyl
gallate, bio-imprinting technique can dramatically enhance the transesterification-catalyzing capability
of tannase. In this work, both cryogenic protection and immobilization were utilized to further improve
its apparent catalytic capability in organic media. The results show that Triton-X-100, mannose, and
magnesium ion all have a positive effect on cryogenic protection of the tannase. Particularly, combina-
tional application of the three cryoprotectants increases its catalytic performance by 2.7-fold factor. Also,
immobilization further elevates its catalytic capability by 2.1 folds. Noteworthily, the coupling application
of immobilization and cryo-protection can cause the conversion rate of substrate of the bio-imprinted
tannase to increase to a promising 70%. Consequently, it will be helpful to fully utilize tannase in organic
phase.
Received 8 February 2013
Received in revised form 12 April 2013
Accepted 16 April 2013
Available online xxx
Keywords:
Cryogenic protection
Immobilization
Bio-imprinting
Tannase
Propyl gallate
Tannic acid
© 2013 Elsevier B.V. All rights reserved.
1
. Introduction
A larger number of enzymes, in general, exhibit obviously lower
and belongs to hydrolysable tannins. Thus, it can be used as a cheap
raw material for production of gallic acid esters, important one of
which is PG. The gallic acid ester has an excellent antioxidative
capacity and, in general, is used in the food industry (particularly
in edible oils or lipid-based food) [9,10]. As a synthetic antioxi-
dant, PG can improve the stability of biodiesel toward oxidation
[11] and also has potential pharmaceutical properties such as an
anti-tumor effect and an antinociceptive activity [12]. Accordingly,
biotransformation of TA to PG owns great economic and ecological
values.
activity in organic medium than in hydrous phase. Improvement
of catalytic capability of biological enzyme in anhydrous media
becomes a crucial question of whether the enzyme is efficiently
utilized in industrial biotransformation. Just as tannase utilized in
synthesis of propyl gallate (PG) from tannic acid (TA) in organic
medium, it has not been applied commercially due to its lower
biocatalytic capability [1,2].
Tannase is a tannin acyl hydrolase (EC 3.1.1.20), that cannot only
hydrolyze the ester and depside linkages in hydrolysable tannins
into glucose and gallic acid in aqueous medium [3] but also catalyze
esterification between gallic acid and alcohol as well as the transes-
terification between TA and gallic acid esters in anhydrous medium
Previously, few studies on tannase-catalyzed transesterification
have been reported since tannase has an undesirable biocatalytic
performance in the transesterification required for PG produc-
tion on commercial scale [1,2]. For this reason, bio-imprinting
technique was introduced to improve its biocatalytic perfor-
mance. The technique, which was firstly defined by Mosbach [13],
can induce a desirable conformation at the active site by pH
tuning [14], substrate or its analogs imprinting [15], and inter-
facial activation [16,17] in an aqueous phase. The conformation
with specific nano-sized cavities can lead to an efficient cou-
pling between enzyme and substrate in organic medium. Whereby,
our group has made combinational use of pH tuning and sub-
strate imprinting to hyper-activate tannase. The modified enzyme
holds 9.7-fold transesterification-catalyzing capability than the
control [18]. Lyophilization, a required step in the protocol of bio-
imprinting, aims to fix the favorable conformation of bio-imprinted
enzyme. As is well-known, enzyme with three dimensional tertiary
[
1,4]. Consequently, tannase has been widespreadly employed in
food and pharmaceutical sectors [5,6].
Tannins are an abundant group of plant secondary metabolites,
which are divided into condensed tannins and hydrolysable tannins
[
7]. TA is composed of a few monomers or dimmers of gallic acid [8]
∗ Corresponding authors. Tel.: +86 551 5593148; fax: +86 551 5593148.
E-mail addresses: zhengzhiming2011@gmail.com (Z. Zheng),
wenjinyue@ahpu.edu.cn (W. Yue).
1
Co-first author, the same contribution to this work as that done by Guangjun
Nie.
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.04.007

2
G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 1–6
structure is so sensitive to temperature variation that mild freezing
can result in the reduction of its biological activity. Hence, cryo-
genic protection of bio-imprinted enzyme is quite essential. But to
our best knowledge, the relevant study has not been seen.
the transesterification-catalyzing capability of the enzyme. C0 and
Ct are the initial concentration of TA and the residual concentra-
tion of TA in a fixed time, t, respectively. In view of C0 and t being
constants, v is proportional to CR. Therefore, CR can be used to
estimate the catalytic activity of the enzyme.
In this present work, combinational cryoprotective technique
(
including three cryoprotectants, such as Triton-X-100, mannose,
The activation factor (AF) refers to the CR of the modified
tannase divided by one of the corresponding control, which intu-
itively denotes the effect of treatment on the catalytic activity of
the enzyme. The control was prepared according to the different
requirements. All experiments were performed in duplicate unless
stated otherwise.
and magnesium ion) was utilized to improve the biocatalytic per-
formance of bio-imprinted tannase. Subsequently, an adsorptive
immobilization was explored to further boost the apparent activ-
ity of the modified tannase. The immobilized and cryoprotective
bio-imprinted tannase (IIt) was used to catalyze the transesteri-
fication from TA to PG. It is endeavored to enhance the catalytic
performance of tannase in organic medium.
2.4. Assay of propyl gallate
2
. Materials and methods
Samples were assayed by HPLC (Waters 600, Waters, USA) with
a Waters 996 photodiode array detector (PDAD) as well as a Pheno-
menex C18 column (250 × 4.60 mm, 4 ␮m). The mobile phase was
composed of 50 mL of methanol, 50 mL of pure water, and 10 ␮L
of acetic acid. The operating temperature was maintained at 35 C.
20 ␮L of sample were injected and were detected at 274 nm with
2.1. Materials
◦
Tannase produced by Aspergillus oryzae was purchased
from Jinan Huazuan Trading Co., Ltd., China. Commercial TA
CB76HB52OB46), citric acid monohydrate, Triton-X-100, mannose,
(
PG (HPLC grade) as the control at a flow rate of 1 mL/min.
magnesium sulfate, celite, n-propanol, and hexane were purchased
from Sinopharm Chemical Reagent Co., Ltd. (SCRC), China, and all
are of an analytical grade. PG (HPLC grade) was purchased from
Sigma Co., USA. All other solvents and reagents were obtained com-
mercially and were of analytical grade.
2.5. Effect of sugar and metal ion on the catalytic capability of
bio-imprinted tannase
Seven types of sugar (including sucrose, lactose, maltose, xylose,
sorbitol, trehalose and mannose) at 100 mM, various concentra-
tions of mannose (0–200 mM), six types of salts including different
melt ions (i.e., manganese chloride, magnesium sulfate, zinc sul-
fate, calcium chloride, copper sulfate, and ferrous chloride) at
2.2. Bio-imprinting, cryogenic protection and immobilization
IIt was prepared based on our preliminary works [18]. This pro-
tocol is subjected to three steps as following:
0
.2 mM, and magnesium ions (0–4.0 mM) were added in IPt solu-
tion, respectively, and then the mixtures were frozen overnight and
lyophilized for 24 h. All of these modified tannases were used to
catalyze the transesterification for synthesis of PG, and the total
amount of TA was unified as 50 mg in all reactions. The effects
of the different treatments were estimated by comparing their CR
values.
Bio-imprinting (modification in aqueous medium): 50 mg TA
was added to the tannase solution, which incorporated 16 IU
(
assayed by rhodanine-spectrophotometric method [19])tannase
with 5 mL 90 mM pH 6.0 citrate buffer as solvent. The uniform
enzyme–substrate mixture was maintained without agitation for
5
min at ambient temperature. The enzyme-substrate mixture was
defined as a TA-pH treated tannase (TPt). (The lyophilized powder
of TPt is considered as bio-imprinted tannase.)
2
.6. Effect of immobilization on the catalytic capability of
bio-imprinted tannase
Cryogenic protection (prior to lyophilization) and immobilization:
1
0
2.5 ␮L 200 mM magnesium ion solution, 0.625 mL 1 M mannose,
.15 mL 10 mM Triton-X-100, and 0.5 g celite were decanted to TPt,
Various doses of celite (0–0.5 g) were added in IPt solution plus
magnesium ions (0.5 mM), 125 mM mannose, and 0.3% Triton-X-
in turn, and then the mixture was agitated for 5 min.
1
00, respectively, and then these mixtures were frozen overnight
Lyophilization: The modified enzyme mixture was frozen at
◦
and lyophilized for 24 h. All of these modified tannases were used
to catalyze the transesterification for synthesis of PG, and the total
amount of TA was unified as 50 mg in all reactions. The effects of the
different treatments were estimated by comparing their CR values.
−
20 C overnight, followed by lyophilization using a freeze dryer
(
VirTis, SP Scientific USA). The lyophilized powders (i.e., IIt) were
◦
stored at 4 C until use.
As for different requirements, the protocol was modified based
on the above steps.
3. Results
2.3. Transesterification synthesis of propyl gallate
3.1. Effect of sugar on the biocatalytic capability of bio-imprinted
tannase
An aliquot of IIt (containing 50 mg TA) was added into
approximate 10 mL reaction medium, which is composed of 1 mL
n-propanol, 9 mL hexane, and 0.1 mL distilled water. The reaction
was performed at 40 C and 200 rpm for 24 h. Conversion rate (CR)
Generally, some sugars were utilized as cryoprotectants toward
◦
protein [20–23]. In this study, the effects of seven sugars on the bio-
catalytic activity of the tannase were analyzed. Fig. 2a shows that
four sugars (i.e., lactose, xylose, sorbitol, and mannose) protect tan-
nase activity better than the three others. Wherein, mannose is the
best protector, by which the enzyme protected obtains the maxi-
mal CR, around 14%. The dependence of the protective effect on the
concentration of mannose was inspected between 5 and 200 mM.
As shown in Fig. 2b, the CR increases with mannose concentra-
tion rising up to 150 mM, at which the maximum CR is 16.3%, 1.6
times that of the control. Conversely, the CR decreases when the
concentration increases further.
in the transesterification reaction refers to the mole percent of TA
completely transformed to PG (C − Ct) relative to the total dose of
0
TA (a fixed concentration, C ) before the equilibrium of reaction (in
0
a fixed time, t). The reaction rate can be calculated as the following:
C − Ct
(C − Ct)C
CR × C
0
0
0
0
v =
=
=
t
tC
t
0
If k is defined as C /t, v = kCR, where v is the reaction rate of
0
transesterification catalyzed by tannase, which, in general, denotes

G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 1–6
3
Table 2
3.2. Effect of metal salt on the biocatalytic capability of
Box–Behnken factorial design and the values of responses.
bio-imprinted tannase
Run
X1
X2
X3
Y
Cryoprotective function of metal ion has been reported [24–27].
To this end, six types of metal salts were studied to determine
their impacts on the biocatalytic activity of tannase. Fig. 2c shows
that the whole of sulfates (including magnesium sulfate, zinc sul-
fate, and copper sulfate) can improve the catalytic performance
of the enzyme better than all of the chlorates. It could be due
to that anions exert different chaotropic and antichatropic effects
on proteins. Among the three sulfates, magnesium sulfate has a
best positive effect on the performance so that the enzyme treated
by it achieves a CR of 15%, 1.4 folds that of the control. Fur-
thermore, the impact of magnesium ion concentration from 0 to
+
1
2
3
4
5
6
7
8
9
0
1
2
−1
−1
+1
+1
0
0
0
0
−1
+1
−1
+1
0
−1
+1
−1
+1
−1
−1
+1
+1
0
0
0
0
0
0
0
0
8.2%
6.6%
17.2%
9.9%
10.4%
9.2%
9.8%
0
−1
+1
−1
+1
−1
−1
+1
+1
0
9.2%
9.2%
1
1
1
13
14
22.7%
10.1%
12.3%
18.7%
17.8%
17.5%
4
.0 mM on the catalytic capability of the tannase was studied,
0
0
0
0
0
0
15
and its result is shown in Fig. 2d. The CR grows with the con-
centration of magnesium ion increasing less than 0.5 mM, but it
begins to decline as the concentration continues to rise. The max-
imum CR of 19%, 1.85 times of the control, was achieved when
the concentration was 0.5 mM. Consequently, it can be seen that
appropriate metal ions can effectively protect the activity of tan-
nase.
X1, X2, and X3 refer to concentration of Triton-X-100, concentration of magnesium
sulfate, and concentration of mannose, respectively.
Table 3
ANOVA for the response.
Source
SS
MS
F
Pr > F
The effects of six surfactants (i.e., SLS, Tween 20, Tween 80,
organosilicon, surfactin and Triton-X-100) on cryogenic protec-
tion of tannase were investigated in preliminary work [18], and
the Triton-X-100 was the best protector. It exhibits the best
performance in term of cryogenic protection of tannase activity
when its concentration is 0.2%. Also, the result showed that all
of the non-ionic surfactants among the six surfactants have bet-
ter cryoprotective effect than SLS, an ionic surfactant. Taking both
surfactant and sugar having hydrophilic groups into account, it is
hypothesized that they probably have a combinational effect on the
cryogenic protection of the enzyme. Therefore, the investigation
of the combinational effects of the different factors is interest-
ing for further improving the biocatalytic performance of the
tannase.
X1
X2
X3
X1X3
X2X2
X3X3
Model
Error
0.6306
0.0852
0.0632
0.1239
0.7285
0.1214
1.7965
0.0778
0.6306
0.0852
0.0632
0.1239
0.7285
0.1214
0.1996
0.0156
40.5346
5.4753
4.0622
7.9639
46.8256
7.8048
12.8306
0.0014
0.0664
0.0999
0.0370
0.0010
0.0383
0.0059
X1, X2, and X3 refer to concentration of Triton-X-100, concentration of magnesium
sulfate, and concentration of mannose, respectively.
Y = 2.8898 + 0.2808X − 0.1032X − 0.0889X − 0.1653X X
1
2
3
1 1
−
−
0.0859X X − 0.1759X X − 0.4442X X + 0.0141X X
1
2
1
3
2
2
2 3
0.1813X X3
(1)
3
3.3. Combinational effects of Triton-X-100, magnesium ion, and
mannose on the biocatalytic capability of bio-imprinted tannase
where Xi and Y refer to the independent variable coded value
and the response (CR), respectively. This model indicates that the
variable with largest effect is the quadratic effect of magnesium
Based on the aforementioned single-factor experiments, a
Box–Behnken factorial experiment was designed to investigate
combinational effects among Triton-X-100, magnesium ion, and
mannose on the biocatalytic activity of tannase. The independent
variables and their coding levels are given in Table 1. Table 2 shows
a three-factor, three-level face-centered cubic design, and their
responses.
2
sulfate (X ). Furthermore, the linear effect of Triton-X-100 and
2
quadratic effect of mannose are more significant than other fac-
tors. Therefore, the surfactant is of great importance in enhancing
the activity of the modified tannase. This effect of the surfactant
can result from combinational function of cryo-genetic protection
together with promoting the enzyme to disperse well in organic
media. Based on Eq. (1), canonical analysis found a stationary point,
where a predicted response with a CR of 22% was acquired under
the optimal factor composition: 0.3% Triton-X-100 (+1), 0.5 mM
magnesium sulfate (0), and 125 mM mannose (−0.5). Verification
experiments gave a CR of 23.3%, in agreement with the predic-
tion. The CR is 2.68-fold that of the control (shown in Fig. 1). It
demonstrates that combinational application of several different
cryoprotectants really protects the hyper-activity of bio-imprinted
tannase.
The analysis of variance (ANOVA) is summarized in Table 3. The
result shows that all of the factors, (i.e., Triton-X-100 concentration,
magnesium sulfate concentration, and mannose concentration)
affect the AF (P < 0.1), and that the surfactant significantly cor-
relates with mannose in enhancing the biocatalytic activity of
the tannase (P < 0.05). Therefore, it is convinced that the surfac-
tant have a similar function to the sugar in cryogenic protection.
The second-order model was employed to fit the response to
2
the independent variables as shown in the following (R 95.85%):
3.4. Effect of immobilization on the biocatalytic capability of
cryoprotective bio-imprinted tannase
Table 1
Real and encoded values for each independent variable for factorial design.
In this present study, it was found that the lyophilized tannases
treated by TA, citric acid, and mannose became more viscous than
the lyophilized powder of the control (non-treated tannase).Under
the condition of high viscosity, the dispersibility of the modified
tannase would weakens in organic media and thus the catalytic
Factor
−1
0.1
0.2
100
0
+1
Concentration of Triton-X-100 (X1)/%
Concentration of magnesium sulfate (X2)/mM
Concentration of mannose (X3)/mM
0.2
0.5
0.3
0.8
150
200

4
G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 1–6
Table 4
Effects of immobilization on the catalytic capabilities of different modified tannases.
Modification
Free
Immobilization
CR2/CR1
CR1/% (mean ± SD)
CR2/% (mean ± SD)
Ma
0.05 ± 0.01
7.4 ± 0.33
18.03 ± 0.1
23.3 ± 2.8
0.9 ± 0.01
1.92 ± 0.1
20.86 ± 1.36
35.87 ± 0.31
49 ± 1.04
38.4
2.82
1.99
2.1
b
M
c
M
d
M
Control
0.40 ± 0.01
0.44
a
M : TA-imprinted tannase, tannase with 16 IU activity and 50 mg TA were uniformly
b
dissolved in 5 mL distilled water. M : pH-tuned tannase, tannase with 16 IU activity
c
was uniformly dissolved in 5 mL 90 mM citrate buffer at pH 6.0. M : The modi-
fied tannase by TA, pH, and Triton-X-100, tannase with 16 IU activity, 50 mg TA,
and 0.15 mL 10 mM Trition-X-100 were uniformly dissolved in 5 mL 90 mM citrate
buffer at pH 6.0. M : Cryoprotective bio-imprinted tannase, 50 mg TA was added
Fig. 1. Scheme of the process of modifying tannase including bio-imprinting, cryo-
genic protection, and immobilization. pH-TA-imprinting: Free tannase was treated
by TA-imprinting and pH tuning (i.e. preparation of lyophilized TPt powder). The
detailed treatment was seen in the part of bio-imprinting presented in Section
d
to the tannase solution, which incorporated 16 IU tannase with 5 mL 90 mM pH 6.0
citrate buffer as solvent. After shaking for about 5 min, 12.5 ␮L 200 mM magne-
sium ion solution, 0.625 mL 1 M mannose and 0.15 mL 10 mM Triton-X-100 were
decanted to the above mixture. Control: Unmodified tannase, tannase with 16 IU
activity was uniformly dissolved in 5 mL distilled water. All the above treatments
2
. Cryogenic protection: TPt was protected against freezing damage. The detailed
d
treatment was seen in that of M in Table 4. Immobilization: identical celite (0.5 g)
was added in free native tannase solution and cryoprotective bio-imprinted tannase
solution, respectively. The other conditions were the same as those given in Section
◦
have four parallels, where two parallels were frozen at −20 C overnight, followed
2.
by lyophilization, and another two parallels were mixed with 0.5 g celite, and then
◦
the mixtures were also frozen at −20 C overnight, followed by lyophilization. The
performance will be reduced. An experiment was designed to verify
the above inference. When the unmodified tannase was immobi-
lized on celite, its catalytic activity diminished. It suggests that the
immobilization itself cannot enhance the activity of tannase. While
TA-imprinted tannase, pH-tuned tannase, TA-pH and Trition-X-
former and the latter refer to free and immobilized enzymes, respectively. The other
conditions were the same as those given in Section 2.
optimal CR of IIt was 49% when it was immobilized on 0.5 g celite,
and the CR is 2.1 times higher than that of the free control (23.3%)
and 123-time that of the original control (0.4%) (shown in Fig. 1).
Accordingly, the adsorptive immobilization has a vigorous ability
to improve the appreciable activity of the modified tannase. The CR
of IIt decreases weakly after it have been continuously recycled for
three times. This suggests IIt is remarkably stable.
1
00 modified tannase, and cryoprotective bio-imprinted tannase
were immobilized, their immobilized ones have 38.4-, 2.8-, 1.9-,
and 2.1-fold enhancements in catalytic performance, respectively
(
(
shown in Table 4). It is certain that the modification processes
i.e., bio-imprinting and cryogenic protection) but not immobiliza-
tion have a remarkable impact on enhancing the real biocatalytic
performance of tannase. From another perspective, the comparison
of these enhancements caused by immobilization indicates that TA
might have the best positive effect on the viscosity of the tannase,
followed by citric acid and mannose. On the contrary, Trition-X-
Based on the treatments of cryogenic protection and immobi-
lization, the bio-imprinted tannase (i.e., IIt) obtained a higher CR
of 70% in the transesterification from TA to PG (shown in Fig. 3a),
and the equilibrium of reaction appeared in 72 h. However, a lower
CR occurs to the reaction catalyzed by native tannase (Nt) through-
out 96 h. The comparison of their initial reaction rates shows that
IIt is 32 folds of Nt (shown in Fig. 3b). This manifests that the
modifications including bio-imprinting, cryogenic protection and
immobilization cannot only obviously increase the CR, but also
strikingly accelerates the process of the reaction.
1
00 could decrease the viscosity and promote substrate accessing
to the active site of the enzyme. The growth of the viscosity could
significantly decrease the dispersibility of the modified tannase
and further decrease their apparent activity. It is demonstrated
that the immobilization can decrease the viscosity and enhance the
apparent catalytic activity of the tannases. Accordingly, the immo-
bilization of the cryoprotective bio-imprinted tannase is greatly
crucial for the improvement in its apparent activity.
4. Discussion
The effect of celite mass on the activity of tannase was inves-
tigated to better the dispersibility of the modified tannase. The
In this work, combinational cryogenic protection has a consid-
erable positive effect on the catalytic performance of bio-imprinted
Fig. 2. Illustration of sugar including its type (a) and mannose concentration (b) as well as metal ion containing its type (c) and magnesium sulfate concentration (d) affecting
the biocatalytic capability of bio-imprinted tannase. The reaction conditions were the same as those given in Section 2. The control CR of a–b and c–d was 10.2% and 10.6%,
separately. The black squares and the blank triangles refer to CR and AF values, respectively.

G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 1–6
5
before lyophilization. Some previous studies also revealed the rein-
forcement effect of magnesium ion on the activity of tannase
[
31,32]. Other studies have shown that certain divalent cation,
notably zinc ion, may enhance the effectiveness of cryoprotec-
tive solutes for stabilizing labile enzymes [22,24,27]. Moreover,
it should be noted that the combinational utilization of metal
ion and carbohydrate may exhibit better their cryoprotective per-
formances than individual application of each. In this work, the
comprehensive application of Triton-X-100, mannose, and magne-
sium ion also can preserve the catalytic capability of IIt well. This
suggests that the combinational cryogenic protection by the cry-
oprotectants really can stand up to various harmful effects from
lyophilization more effectively than a single cryogenic protection
by each one. This finding has been supported by some previous
studies on other proteins, i.e., the biological activity of the PFK
was preserved by exposing the protein to a trehalose and zinc
ion prior to freezing [22]. The interaction of sucrose and zinc for
cryogenic protection of fish actomyosin has been well investigated
[25].
It is troublesome that enzyme molecules, which easily dis-
solve in aqueous solution, converge with each other in anhydrous
phase. Moreover, a few hydrophilic adapting factors, such as TA,
citric acid, and mannose, can intensify the convergence (particu-
larly in anhydrous media). Therefore, it is of great significance to
immobilize the cryoprotective bio-imprinted tannase for improv-
ing its dispersibility in anhydrous phase. As for the cryoprotective
bio-imprinted tannase, a simple adsorptive immobilization is com-
petent to amplify its appreciable activity in water-immiscible
medium. Upon being immobilized, the enzyme can be conveniently
separated from the reaction medium, and this is beneficial for its
recycling. In addition, celite, as an economical support, is helpful
to an industrial application. The modifications based on cryo-
genic protection and immobilization can dramatically promote the
biocatalytic performance of the bio-imprinted tannase in organic
solvent and make its catalytic capability increased by 5.6 folds. The
consequence suggests that bio-imprinting technique coupled with
other modifications like cryogenic protection and immobilization
just make bio-imprinted enzyme exhibit its real catalytic activity.
Notably, the immobilized and cryoprotective bio-imprinted tan-
nase biocatalyst achieves a promising CR of 70%, evidently higher
than the latest reports. Where, a synthetic yield of 45% catalyzed
by an immobilized tannase was obtained in hydrous medium com-
posed of 30% of 1-propanol and 70% water [1]. If with concentrated
sulfuric acid as catalyzer, the CR of transesterification was appro-
priate 50% [33]. Taking gallic acid as substrate, the esterification
catalyzed by mycelium-bound tannase from Aspergillus niger in
benzene just had a CR of 65% [4].
Fig. 3. Comparison of the immobilized and cryoprotective bio-imprinted tannase
and native tannase in reaction process (a) and in initial reaction rate (b). IIt is the
immobilized and cryoprotective lyophilized powder of TPt. Its preparation was seen
in Section 2. Where, the pH value of citrate buffer was changed as 5.0. Nt is a native
tannase without any treatments. Aliquots of IIt and Nt were used to catalyze the
transesterification in an organic medium consisted of 1.5 mL n-propanol, 150 ␮L of
◦
9
0 mM pH 5.0 citrate buffer, and 18.5 mL hexane at 40 C and 200 rpm. The total
amount of TA was unified as 50 mg in all reactions. Other reaction conditions were
the same as those given in Section 2.
tannase. With regard to surfactant, non-ionic surfactants like
Triton-X-100, in general, protect the activity of tannase better than
ionic surfactants, and their concentrations usually are below criti-
cal micelle concentration (CMC) [16,28] because their hydrophobic
ends may competitively bind the active site of enzyme against its
substrate under the condition of excessive surfactant [28]. Other-
wise, surfactant not only enhances protein rigidity and substrate
accessibility to the active site but also induces a more competent
catalytic center in anhydrous media [16,17,28].
The result presented in this work also indicates that Triton-
X-100 has a correlation with mannose in cryogenic protection
of the catalytic capability of the bio-imprinted tannase possibly
because they both have hydrophilic groups that bind to the sur-
face of tannase competitively. Furthermore, mannose as a filler
maybe provides an effective support structure for tannase so
that the structure of carbohydrate-bound protein can resist the
deleterious impacts of crystallization during slow freezing. On
the other hand, sugar molecules with several hydroxyl groups
can prevent enzyme from dehydration-caused damage by replac-
ing water molecules around the proteins during lyophilization,
and an excipient-induced activation can lead to an increment of
the activity as well [20]. With regard to different enzymes, the
cryoprotective effect of the sugar may be different. Carpenter
et al. [22] reported that trehalose was a perfect cryoprotective
agent for phosphofructokinase (PFK). Another team convinced that
the combinational use of glucose and glycerol protected lipo-
some more efficiently than the application of other carbohydrates
such as mannose [29]. In addition, glucose, sucrose, and raffinose
were used as cryoprotectants to preserve chloroplast thylakoids
5. Conclusions
In summary, the combinational application of cryogenic protec-
tion and simple immobilization can not only improve the apparent
catalytic performance of bio-imprinted tannase, but also obviously
accelerate its reaction process. Accordingly, this present work
might present a reference for enhancing the apparent activity of
bio-imprinted enzyme in organic media and broaden its application
scope.
[
30]. It was proposed that branched oligosaccharides can protect
bovine serum albumin (BSA) by preserving BSA in a rigid structure
21].
It has been reported that metal ions play a role in cryogenic
Acknowledgements
[
This work has been supported by grants from the Knowl-
edge Innovation Program of the Chinese Academy of Sciences
protecting biological activity of protein [24,25,27]. In this present
research, magnesium ions dramatically enhance the catalytic capa-
bility of the bio-imprinted tannase. This may be ascribed to the
interaction of magnesium ion(s) with the active site of tannase.
As a result, a preferred active conformation of enzyme is induced
(KSCX2-YW-G-050), the National High Technology Research and
Development Program of China (2009AA02Z305), and National
Nature Science Foundation (51202002, 31171753).

6
G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 1–6
References
[16] I. Mingarro, C. Abad, L. Braco, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 3308–3312.
[
17] N. Kamiya, H. Kasagi, M. Inoue, K. Kusunoki, M. Goto, Biotechnol. Bioeng. 65
1999) 227–232.
18] G.J. Nie, Z.M. Zheng, W. Jin, G.H. Gong, L. Wang, J. Mol. Catal. B: Enzym. 78 (2012)
2–37.
(
[
[
[
[
[
1] G. Fernandez-Lorente, J.M. Bolivar, J. Rocha-Martin, J.A. Curiel, R. Mu n˜ oz, B. de
las Rivas, A.V. Carrascosa, J.M. Guisan, Food Chem. 128 (2011) 214–217.
2] A. Gaathon, Z. Gross, M. Rozhanski, Enzyme Microb. Technol. 11 (1989)
[
3
[19] S. Sharma, T.K. Bhat, R.K. Dawra, Anal. Biochem. 279 (2000) 85–89.
[20] K. Debulis, A.M. Klibanov, Biotechnol. Bioeng. 41 (1993) 566–571.
[21] J. Auh, Y. Kim, P. Cornillon, J. Yoon, S. Yoo, K. Park, Int. J. Food Sci. Technol. 38
6
04–609.
3] B. Mahendran, N. Raman, D.J. Kim, Appl. Microbiol. Biotechnol. 70 (2006)
44–450.
4] X.W. Yu, Y.Q. Li, S.M. Zhou, Y.Y. Zheng, World J. Microbiol. Biotechnol. 23 (2007)
091–1098.
4
(
2003) 553–563.
[
22] J.F. Carpenter, S.C. Hand, J.H. Crowe, L.M. Crowe, Cryogenic protectant for
proteins. University of Southwestern Louisiana, United States Patent, patent
number: 4,806,343, Application number: 839,330 1989.
1
5] M. Chávez-González, L.V. Rodríguez-Durán, N. Balagurusamy, A. Prado-
Barragán, R. Rodríguez, J.C. Contreras, C.N. Aguilar, Food Bioprocess. Technol. 5
[
[
[
[
23] M.C. Heller, J.F. Carpenter, T.W. Randolph, Biotechnol. Bioeng. 63 (1999)
(
2011) 445–459.
1
66–174.
24] K.C. Hazen, L.D. Bourgeois, J.F. Carpenter, Arch. Biochem. Biophys. 267 (1988)
63–371.
25] G.A. MacDonald, T.C. Lanier, F.G. Giesbrecht, J. Agric. Food Chem. 44 (1996)
13–118.
26] J.F. Carpenter, S.C. Hand, L.M. Crowe, J.H. Crowe, Arch. Biochem. Biophys. 250
1986) 505–512.
[
[
6] C.N. Aguilar, R. Rodriguez, G. Gutierrez-Sanchez, C. Augur, E. Favela-Torres, L.A.
Prado-Barragan, A. Ramirez-Coronel, J.C. Contreras-Esquivel, Appl. Microbiol.
Biotechnol. 76 (2007) 47–59.
3
7] B. Adamczyk, S. Adamczyk, A. Smolander, V. Kitunen, Soil Biol. Biochem. 43
1
(
2011) 628–637.
[
[
8] S.C. Chen, K.T. Chung, Food Chem. Toxicol. 38 (2000) 1–5.
9] S. Noel Robledo, M. Alicia Zón, C. Daniel Ceballos, H. Fernández, Food Chem.
(
[
[
27] J.F. Carpenter, S.C. Hand, L.M. Crowe, J.H. Crowe, Google Patents, 1990.
28] G. Fernandez-Lorente, J.M. Palomo, Z. Cabrera, R. Fernandez-Lafuente, J.M.
Guisan, Biotechnol. Bioeng. 97 (2007) 242–250.
1
27 (2011) 1361–1369.
[10] C. Jacobsen, K. Hartvigsen, P. Lund, A.S. Meyer, J. Adler-Nissen, J. Holstborg, G.
Holmer, Eur. Food Res. Technol. 210 (1999) 13–30.
[29] B. Stark, G. Pabst, R. Prassl, Eur. J. Pharm. Sci. 41 (2010) 546–555.
[30] R.D. Lineberger, P.L. Steponkus, Plant Physiol. 65 (1980) 298.
[31] Y. Anwar, I. Artika, H. Danuri, Hayati J. Biosci. 16 (2010) 95–99.
[32] V. Chhokar, M. Sangwan, V. Beniwal, K. Nehra, K.S. Nehra, Biotechnol. Bioeng.
[
[
[
11] A. Sarin, N.P. Singh, R. Sarin, R.K. Malhotra, Energy 35 (2010)4645–4648.
12] H.J. Jung, C.J. Lim, Phytother. Res. 25 (2011) 1570–1573.
13] M. Stahl, U. Jeppssonwistrand, M.O. Mansson, K. Mosbach, J. Am. Chem. Soc.
1
13 (1991) 9366–9368.
160 (2010) 2256–2264.
[
[
14] M.N. Gupta, I. Roy, Eur. J. Biochem. 271 (2004) 2575–2583.
15] A. Fishman, U. Cogan, J. Mol. Catal. B: Enzym. 22 (2003) 193–202.
[
33] G. Wang, Q. Chen, W. Gu, J. Cent. South Univ. Forest Technol. 30 (2010) 137–140.