Development of a tannase biocatalyst based on bio-imprinting for the production of propyl gallate by transesterification in organic media

Article abstract of DOI:10.1016/j.molcatb.2012.01.007

A bio-imprinting technique was applied to activate tannase in order to enhance its biocatalytic activity. Specifically, the effects of three bio-imprinting methods (i.e. substrate imprinting, pH imprinting, and interfacial imprinting) on the activating factor of tannase were investigated. The results show that bio-imprinting methods can activate tannase remarkably, and they were combined to develop a tannase biocatalyst with a 40% conversion rate of substrate, 100-fold higher than that of the control. This approach can be used to construct an effective way to produce propyl gallate as well as to exploit readily available tannic acid. The immobilized bio-imprinted tannase can catalyze the synthesis of propyl gallate from tannic acid by transesterification in organic media. This work not only presents an effective means of making use of various tannic acid-rich agro-forestry residues, but also broadens the field of applications of the bio-imprinting technique.

Full text of DOI:10.1016/j.molcatb.2012.01.007

Journal of Molecular Catalysis B: Enzymatic 78 (2012) 32–37
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Development of a tannase biocatalyst based on bio-imprinting for the production
of propyl gallate by transesterification in organic media
Guangjun Niea,b,c, Zhiming Zhenga,∗, Wei Jina, Guohong Gonga, Li Wanga
a Key Lab of Ion Beam Bioengineering, Chinese Academy of Science, 230031 Hefei, China
b School of Life Science, University of Science and Technology of China, 230022 Hefei, China
c College of Biochemical Engineering, Anhui Polytechnic University, 241000 Wuhu, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A bio-imprinting technique was applied to activate tannase in order to enhance its biocatalytic activity.
Specifically, the effects of three bio-imprinting methods (i.e. substrate imprinting, pH imprinting, and
interfacial imprinting) on the activating factor of tannase were investigated. The results show that bio-
imprinting methods can activate tannase remarkably, and they were combined to develop a tannase
biocatalyst with a 40% conversion rate of substrate, 100-fold higher than that of the control. This approach
can be used to construct an effective way to produce propyl gallate as well as to exploit readily available
tannic acid. The immobilized bio-imprinted tannase can catalyze the synthesis of propyl gallate from
tannic acid by transesterification in organic media. This work not only presents an effective means of
making use of various tannic acid-rich agro-forestry residues, but also broadens the field of applications
of the bio-imprinting technique.
Received 14 September 2011
Received in revised form 5 January 2012
Accepted 6 January 2012
Available online 16 January 2012
Keywords:
Bio-imprinting
Propyl gallate
Tannase
Tannic acid
© 2012 Elsevier B.V. All rights reserved.
Transesterification
1. Introduction
is a specific substrate of tannase. The conversions of TA to gallic acid
[12] or propyl gallate [13] by tannase have recently been reported.
Enzymes are well recognized as practical biocatalysts of
immense potential and, in particular, are being increasingly used
on an industrial scale for bio-transformation [1], preparation of fine
chemicals, and synthesis of enantiopure pharmaceuticals [2–4]. In
the future, more and more biological resources will be transformed
into high value-added products by biocatalysts. These biocatalytic
syntheses will replace conventional chemical syntheses, which
often employ corrosive reagents and require high energy input.
Propyl gallate (PG) is a very important gallic acid ester usually
used as an antioxidant in foods, cosmetics, hair products, adhe-
sives, and the lubricants industry [5,6]. It has also been the focus
of attention because of its possible pharmaceutical applications
[7–10]. Most available propyl gallate is synthesized mainly from
gallic acid by the conventional chemical method, which uses con-
centrated sulfuric acid as a chemical catalyst. It would be preferable
to replace the chemical synthesis with a green biocatalytic method.
Tannase, tannin acyl hydrolase (E.C. 3.1.1.20), can not only
hydrolyze the ester and depside bonds in tannic acid (TA) to release
gallic acid and glucose, but can also catalyze the transesterification
or esterification syntheses of gallic acid esters [11] in anhydrous
media. Due to the presence of many phenolic hydroxyl groups, TA
The bioconversion of TA to propyl gallate (shown in Scheme 1)
[13,14] entails fewer procedures and lower cost than the con-
ventional chemical synthesis. However, the biocatalytic activity of
natural tannase in the transesterification is so low that the conver-
sion ratio of substrate (CR) prevents it from being a viable industrial
application. To improve the CR, most previous studies have focused
on the immobilization of tannase [12,13,15]. Notwithstanding, the
CR of immobilized tannase is still restricted by the limitations of
the natural enzyme structure.
A bio-imprinting technique is, in general, based on modifying
the conformation of an enzyme by changing the pH or by adding
substrate or corresponding analogs, surfactants, and other enti-
ties to the aqueous phase. The aim is to make the enzyme adopt
a conformation with specific nano-sized cavities that enable pre-
cise coupling between enzyme and substrate. These cavities can
be retained when the enzyme is transferred from aqueous media
to anhydrous media due to their dynamical rigidity in the organic
phase [16]. An imprinted enzyme may have high catalytic speci-
ficity, affinity, and stability [17]. Therefore, the technique can break
through limitations in the natural enzyme structure. Thus far,
bio-imprinting techniques have been used to improve several bio-
catalysts. For example, the biocatalytic activities of chymotrypsin
[2] and lipase [18–20] were improved based on bio-imprinting
with the substrate (i.e. substrate imprinting). Lipase was also mod-
ified based on bio-imprinting with the surfactant (i.e. interfacial
∗
Corresponding author. Tel.: +86 551 5593148; fax: +86 551 5593148.
E-mail address: zmzheng@ipp.ac.cn (Z. Zheng).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2012.01.007

G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 32–37
33
Scheme 1. Transesterification reaction to form propyl gallate from tannic acid.
imprinting) [4,21–23]. An enzyme with a ‘pH memory’ can be acti-
vated by pH tuning (i.e. pH imprinting). Furthermore, the strategy of
combinational imprinting has also been applied to enhance the cat-
alytic performance of lipase [24,25]. These improved biocatalysts
exhibit surprisingly high activities compared to the corresponding
controls. However, improvements in tannase activity based on a
bio-imprinting technique have not been reported previously.
In this study, the bio-imprinting technique has been used to
enhance the transesterification activity of tannase. Three bio-
imprinting methods (i.e. substrate imprinting, pH imprinting, and
interfacial imprinting) were investigated to develop a promising
biocatalyst that could be used to synthesize propyl gallate from TA
in organic media. This report explores an effective way to biotrans-
form TA coupled with a practical method for synthesis of propyl
gallate on an industrial scale.
at 40 ◦C and 200 rpm for 24 h. CR is the mole ratio of 10% propyl
gallate formed to TA in the initial reaction mixture.
2.4. Assay of propyl gallate
Samples were analyzed by HPLC (Waters 600, Waters, USA)
equipped with a Waters 996 photodiode array detector (PDAD) and
Phenomenex C18 column (250 mm × 4.60 mm, 4 ␮m). The mobile
phase consisted of 50 mL of methanol, 50 mL of water, and 10 ␮L
of acetic acid. The operating temperature was maintained at 35 ◦C.
20 ␮L of sample were injected and peaks were detected at 274 nm
with propyl gallate as the control at a flow rate of 1 mL/min. Sam-
ple preparation was subjected to the following protocol: (1) 200 ␮L
reaction solution were diluted 10-fold with methyl alcohol (HPLC
grade), (2) the diluents were filtered using an organic membrane
with a pore size of 0.45 ␮m. All experiments were performed in
duplicate unless stated otherwise.
2. Materials and methods
3. Results and discussion
2.1. Reagents
3.1. Effect of tannic acid on the activity of imprinted tannase
Tannase was purchased from Jinan Huazuan trading Co.,
Ltd., China. Commercial TA (C76H52O46); GA monohydrate
(C7H6O5·H2O), Triton X-100, citric acid monohydrate, and celite
were purchased from Sinopharm Chemical Reagent Co., Ltd. (SCRC),
China. TA, GA, and citric acid were of analytical grade. All other
solvents and reagents were obtained commercially and were of
analytical grade.
Fig. 1 shows that the TA-imprinted tannase (TE) has a lower CR
than the control (non-imprinted tannase with distilled water as
solvent, Ew). However, when TE and Ew were individually fixed on
the celite, the immobilized TE (TEi) achieved a 4.86-fold enhance-
ment in CR compared to the immobilized Ew (Ei). When citric acid
buffer replaced the distilled water in the TE solution, the buffer-
TA-imprinted tannase (BTE) attained a CR of 7.56%, approximately
150-fold higher than that of TE. Further experiments showed that
TA addition results in a steep reduction in the pH value of distilled
water, whereas it hardly changes the pH value of the citric acid
buffer (data not shown). The effect of the quantity of TA added on
the AF of tannase is shown in Fig. 2a. When the mass of TA was
below 0.04 g, the AF increased with TA mass. When the mass of TA
was 0.04 g, the imprinted tannase achieved a CR of 5.7% and an AF
of 1.4.
Comparison of the CR values of TE and Ew indicates that TA
appears to inactivate tannase. As shown by further experiments,
TA inhibits the biocatalytic activity of tannase by changing the
pH value in the hydrated shell around the enzyme. In contrast,
comparison of the CR values of immobilized TE and immobilized
Ew implies that TA can activate immobilized tannase, significantly
enhancing the biocatalytic activity of TE by reducing molecular
aggregation. The aggregation results from the interaction of TA with
free tannase. The difference in CR between BTE and TE suggests
that more efficient substrate imprinting may require a favorable
conformation modulated by pH imprinting. Fig. 2a indicates that
TA substantially improves the biocatalytic activity of tannase, and
the AF of tannase is affected by the TA concentration. This may
2.2. Bio-imprinting
50 mg TA and 16 IU tannase were uniformly dissolved in 5 mL
of 90 mM citrate acid buffer at pH 6.0, followed by the addition of
0.15 mL of 0.3% Triton X-100 surfactant and 0.5 g celite, and the
mixture was frozen at −20 ◦C overnight. Finally, the sample was
lyophilized in a freeze dryer (VirTis, SP Scientific USA) to a pow-
der, and then stored at −20 ◦C until use. The bio-imprinting process
and parameters were the same throughout unless stated otherwise.
The activating factor (AF) refers to the enhancement in CR of the
bio-imprinted tannase relative to the corresponding control, and
it is calculated as the ratio of the CR of imprinted tannase to that
of the control. The controls were prepared according to different
requirements.
2.3. Transesterification synthesis of propyl gallate
An aliquot of imprinted biocatalyst containing 0.05 g TA was
added to a 25-mL flask with approximately 10 mL of solvent mix-
ture composed of 1 mL of n-propanol, 9 mL of hexane, and 100 ␮L
of distilled water. The transesterification reaction was conducted

34
G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 32–37
Fig. 3. Schematic diagram of the three bio-imprinting processes. (a) Natural tan-
nase, (b) pH-imprinted tannase, (c) pH- and surfactant-imprinted tannase, (d)
combinational bio-imprinted tannase with pH-, surfactant- and TA-imprinting
and (e) unloaded bio-imprinted tannase with high biocatalytic activity. Step 1:
pH-imprinting step, the molecular conformation of natural tannase changes with
variations in the ionic state of tannase caused by bio-imprinting with 90 mM pH 5.5
citric acid buffer. Step 2: Interfacial-imprinting step, binding of surfactant to tan-
nase further stabilizes the molecular conformation of buffer-imprinted tannase, and
improves the interfacial qualities of the enzyme. Step 3: Substrate-imprinting step,
the specific catalytic site of tannase and the transition complex of enzyme–substrate
are introduced with coupling of TA and tannase. Step 4: The removal step, unloaded
complex bio-imprinted tannase is formed through removal of all ligands in organic
solution.
Fig. 1. Comparison of the effects of different treatments on the CR of tannase. Ew:
The non-imprinted tannase, 16 IU tannase were uniformly dissolved in 5 mL distilled
water, and then the solution was frozen overnight and lyophilized for 24 h. TE: The
TA-imprinted tannase, 16 IU tannase and 0.05 g TA were uniformly dissolved in 5 mL
distilled water, and then the mixture was frozen overnight and lyophilized for 24 h.
BTE: The buffer- and TA-imprinted tannase, 16 IU tannase and 0.05 g TA were uni-
formly dissolved in 5 mL 50 mM pH 5.5 citric acid buffer, and then the mixture was
frozen overnight and lyophilized for 24 h. Ei and TEi are Ew and TE immobilized on
celite, respectively. These tannases were applied to catalyze the synthesis of propyl
gallate by transesterification. The effects of different treatments on the biocatalytic
activity of tannase have been estimated by comparing the CRs. The catalytic reaction
conditions were the same as those given in Section 2.
and thus the apparent activity is reduced. Previous studies have
reported that some enzymes are hyper activated by a substrate
analog on the basis of bio-imprinting [20,27–29]. In addition, some
researchers [18–20] have directly used the substrate as the imprint-
ing template. However, to our knowledge, substrate imprinting
has not been reported before as a means of improving the bio-
catalytic activity of tannase. From the above discussion, it appears
that TA can activate tannase significantly. At the same time, it
meets the requirements (i.e. resemblance to substrate or com-
petitive inhibitor and solubility in the aqueous phase [29]) of a
favorable imprinting template. If TA can be used as the template in
bio-imprinting, the step of removing the bio-imprinting template
(shown in step 4 in Fig. 3) is avoided. This not only simplifies the
imprinting process, but also decreases the loss of enzyme result-
ing from the removal step. Therefore, substrate imprinting is very
suitable for industrial applications.
3.2. Effect of pH on the activity of imprinted tannase
Fig. 2b shows that the AF increases as the concentration of cit-
ric acid buffer increases from 0 to 50 mM, reaches a maximum at
50 mM, and then decreases slowly after 50 mM. The AF increases
when the pH value of the citric acid buffer is below 5.5, at which
it reaches a maximum (shown in Fig. 2c). This suggests that the
AF significantly depends on the pH value and the buffer concen-
tration. Previous studies have reported that some enzymes were
hyper-activated by changing the pH value in the aqueous microen-
vironment around the enzyme [14,16]. In this work, the optimal pH
value of tannase ranges from 4 to 6, and when pH value of the citric
acid buffer was 5.5, the protein molecules may be assembled into a
favorable conformation with high activity. This favorable structure
may also be ascribed to modulation of the ionic state of the enzyme
caused by variation of the buffer concentration. When the enzyme
is transferred into an anhydrous medium from an aqueous solution,
this structure is memorized due to its rigidity in the organic phase
(shown in step 1 in Fig. 3).
Fig. 2. Effects of TA and pH on the biocatalytic activity of tannase. (a) With 50 mM
pH 5.5 citric acid buffer as solvent, 16 IU tannase were imprinted with 0.005–0.05 g
TA, and the control CR was 4%. (b) With 0.04 g TA as template, 16 IU tannase were
imprinted with 5 mL 0–200 mM citric acid at pH 5.5, and the control CR was 0.9%.
(c) With 0.04 g TA as template, 16 IU tannase were imprinted with 5 mL pH 4.5–6.5
citric acid at a concentration of 50 mM, and the control CR was also 0.9%. Substrate
amounts in each test were 0.05 g in the catalytic reaction system. All of the tannases
were applied to catalyze the synthesis of propyl gallate by transesterification. The
effects of TA and pH on the biocatalytic activity of tannase were estimated by com-
paring their CRs and AFs. The catalytic reaction conditions were the same as listed in
Section 2. Squares and triangles refer to CR and AF, respectively (the same as below).
be because the coupling of TA with tannase induces the enzyme
to form an efficient and specialized active center with the assis-
tance of pH imprinting, and to construct a transition complex of
substrate-enzyme at the appropriate concentration of TA (shown
in step 3 in Fig. 3) [26]. However, as the concentration of TA
increases, TE becomes so ‘sticky’ that it prevents diffusion of the
substrate and product into and out of the complex, respectively,
To obtain the optimal AF for TA imprinting together with pH
imprinting, Box–Behnken factorial design was applied on the basis
of the single-factor experiments aforementioned. Table 1 shows
the independent variables and their coding levels. Table 2 presents

G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 32–37
35
Table 1
Defining variables and coding.
Factor
−1
0
+1
TA mass, X1 (g)
Buffer concentration, X2 (mM)
pH value, X3
0.01
20
5
0.03
70
5.5
0.05
120
6
Table 2
Box–Behnken factorial design and the values of responses.
RUN
X1
X2
X3
Y
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
−1
−1
0
0
0
1.98
2.35
0.31
1.79
0.36
1.19
1.67
5.21
3.45
0.75
3.63
7.54
4.02
4.61
4.16
−1
1
−1
1
1
1
0
0
0
0
0
−1
−1
1
1
0
0
0
0
0
−1
1
−1
1
−1
−1
1
1
0
0
0
Fig. 4. Effect of the imprinting temperature on the biocatalytic activity of tannase.
With 90 mM pH 6.0 citric acid buffer as solvent, 16 IU tannase were imprinted with
0.05 g TA at 0–40 ◦C. (a) The imprinted tannase catalyzed the synthesis of propyl
gallate from TA by transesterification. (b) The imprinted tannase catalyzed the syn-
thesis of propyl gallate from gallic acid by esterification. The effect of imprinting
temperature on the biocatalytic activity of tannase was estimated by comparing the
relevant CRs and AFs. The catalytic reaction conditions were the same as those in
Section 2.
−1
1
−1
1
0
0
0
0
0
Table 3
ANOVA for the responses.
Source
SS
1.0629
3.0522
2.752
0.6302
1.2736
3.4953
12.3779
0.376
MS
F
Pr > F
X1
X2
X3
X1*X2
X1*X3
X2*X2
Model
Error
1.0629
3.0522
2.752
0.6302
1.2736
3.4953
1.3753
0.0752
14.1342
40.5883
36.5967
8.38
16.9361
46.4817
18.2893
0.0132
0.0014
0.0018
0.034
0.0092
0.001
0.0026
the three factors and the three-level face-centered cube design, and
the responses. Analysis of variance (ANOVA) shows that all factors
(i.e. TA, buffer concentration, and buffer pH value) have a dramatic
effect on the AF of tannase. Table 3 shows that TA imprinting sig-
nificantly correlates with pH imprinting. This further proves that
TA imprinting activates tannase more when it is coupled with pH
imprinting. A second-order model was used to fit the response to
the independent variables, and the model is shown as the following
(R2 97.05%):
Fig. 5. Effect of surfactant on the biocatalytic activity of tannase. (a) With 90 mM
pH 6.0 citric acid buffer as solvent, 16 IU tannase were imprinted with six types of
surfactants at 0.2%. (b) With 90 mM pH 6.0 citric acid buffer as solvent, 16 IU tannase
were imprinted with 0–0.5% Triton X-100. The imprinted tannases were applied to
catalyze the synthesis of propyl gallate from TA by transesterification. The effect of
surfactant on the biocatalytic activity of tannase was estimated by comparing the
CRs or AF values. The catalytic reaction conditions were the same as those in Section
2, and the control CR was 9.7%.
Y = 1.448342 − 0.364497 ∗ X1 + 0.617674 ∗ X2 + 0.586515 ∗ X3
−0.236851 ∗ X1 ∗ X1 + 0.396913 ∗ X1 ∗ X2
+ 0.564261 ∗ X1 ∗ X3 − 0.972962 ∗ X2 ∗ X2
− 0.014462 ∗ X2 ∗ X3 − 0.146454 ∗ X3 ∗ X3
(1)
where Xi refers to the independent variable coded value and Y is the
response (CR). Based on Eq. (1), the optimal CR of imprinted tan-
nase was predicted to be 8.2%, and the optimal imprinting medium
composition included 0.05 g TA and 5 mL 90 mM pH 6.0 citric acid
buffer. Verification experiments yielded a CR of 8.7% and an AF of
9.7, in agreement with the prediction.
important in enhancing the AF of tannase. In fact, bio-imprinting is
a process correlated with temperature because bio-imprinting is,
in principle, based on molecular thermal motion. At high tempera-
ture the motion becomes too rapid to form the transition complex
of tannase-TA efficiently, whereas at low temperature the motion
is too slow to form an effective coupling of TA with tannase. It has
also been reported that, if the imprinting temperature is too high, it
has a negative impact on the biocatalytic activity of bio-imprinted
lipase [19]. Therefore, it is believed that the proper imprinting
temperature really does contribute to an improvement in CR of
tannase.
3.3. Effect of temperature on the activity of imprinted tannase
The effect of temperature on the AF of tannase is shown in
Fig. 4. In Fig. 4a, the highest CR, 11.81%, occurs at 25 ◦C, and it is
13-fold higher than that of Ew. The same result is seen in Fig. 5b.
This demonstrates that a suitable imprinting temperature is very

36
G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 32–37
Table 4
(BTS); (2) first pH imprinting, second interfacial imprinting, third
substrate (TA) imprinting (BST). Table 4 shows that the immobi-
lized tannase imprinted in the BST order has a slight advantage
in CR over the immobilized tannase imprinted in the BTS order.
This may be due to the fact that TA as the imprinting template
is hydrolyzed to GA by tannase, and thus the improvement in AF
caused by TA imprinting may be reduced. Therefore, once substrate
imprinting is combined with other imprinting methods (i.e. inter-
facial imprinting and pH imprinting), the imprinting order has to
be carefully considered to achieve amplification of the AF.
Effect of the immobilization on the catalytic activity of bio-imprinted tannase.
Treatment
Solvent
Immobilization
CR (mean SD)/%
AF
BE
Ew
SE
BTSE
Es
BEi
BTSEi
BSTEi
Ei
Buffer
F
F
F
F
F
T
T
T
T
7.4 0.33
0.9 0.01
8.22
1.00
1.35
19.39
1.00
52.15
89.68
100.40
1.00
Distilled water
Distilled water
Buffer
Distilled water
Buffer
Buffer
Buffer
Distilled water
1.26 0.07
18.03 0.1
0.93 0.04
20.86 1.36
35.87 0.31
40.16 0.14
0.40 0.01
BE: Buffer imprinted tannase, 16 IU tannase imprinted with buffer. SE: Surfactant
imprinted tannase, 16 IU tannase imprinted with 0.2% Triton X-100 with distilled
water as solvent. BTSE: Combinational imprinted tannase in the order BTS; with
buffer as solvent 16 IU tannase complexly imprinted with 0.05 g TA and 0.2% Triton
X-100. BSTE: Combinational imprinted tannase in the order of BST; with buffer as
solvent 16 IU tannase complexly imprinted with 0.2% Triton X-100 and 0.05 g TA.
Es: The control of SE, a mixture of the tannase lyophilization powder with 0.2%
Triton X-100. BEi, BSTEi, BTSEi, and Ei refer to the immobilized BE, the immobilized
BSTE, the immobilized BTSE, and the immobilized Es, respectively. The imprinted
tannases catalyzed the synthesis of propyl gallate from TA by transesterification.
The effect of immobilization on the biocatalytic activity of tannase was estimated
by comparing the CRs or AFs. The catalytic reaction conditions were the same as
those in Section 2, and all buffers were 90 mM pH 5.5–6.0 citric acid buffer. “F” and
“T” in the “Immobilization” column refer to free enzyme and immobilized enzyme
on 0.5 g celite, respectively.
3.6. Effect of enzyme immobilization on the activity of imprinted
tannase
The effect of enzyme immobilization on CR is shown in Table 4.
It is expected that all forms of the immobilized imprinted tannase
are more active than their free counterparts. In particular, immo-
bilized tannase imprinted in the BST order exhibits a surprising CR
of 40.16%, 100.4-fold that of the original control (the immobilized
Ew). This indicates that just a simple immobilization method can
boost the apparent AF of bio-imprinted tannase remarkably due to
the surface extension of the immobilized enzyme.
Fig. 1 and Table 4 show that immobilized TE, immobilized
BE, and immobilized tannase imprinted in the BTS order Exhibit
4.86-fold, 2.82-fold, and 1.99-fold higher CR, respectively, than
their free counterparts. From this it is inferred that the viscosity
of the imprinted enzyme is higher, and the enzyme immobiliza-
tion improves the apparent activity of the imprinted enzyme by
reducing the viscosity. On the other hand, it verifies once again
that TA in TE can make TE so sticky that mutual agglomeration
occurs, whereas surfactant linked with protein can improve the dis-
persibility of the enzyme sufficiently so as to decrease this effect of
immobilization on the activation of the enzyme.
Furthermore, the imprinted enzyme, unlike natural enzyme, is
usually used in organic media, in which an immobilized enzyme
formed by a simple method such as adsorptive immobilization can
avoid a loss of enzyme activity. In addition, celite has been fre-
quently used as an economical support. Therefore, it is easy to
manufacture an immobilized imprinted tannase with specific and
high biocatalytic activity in an industrial process.
In the present work, a bio-imprinting technique combined with
a simple adsorptive immobilization has been shown to improve
the CR of natural tannase by 40%. As expected, the CR can be fur-
ther enhanced by some other optimizations in the reaction system
and in the reaction conditions. This promising biocatalyst can be
applied to catalyze the transesterification reaction of TA to propyl
gallate. TA is a hydrolysable, water-soluble gallotannin, and is found
in a variety of agro-forestry residues such as wood, bark, leaves,
fruits, and galls [31]. Thus, it is of great importance to the economy
and in environmental protection to synthesize propyl gallate in a
way that couples it with making high-value use of TA. In addition,
the promising biocatalyst is only used in organic media, where the
transesterification reaction occurs more easily than the hydrolysis
reaction. The differences in hydrophobicities of propyl gallate, sub-
strate, and biocatalyst allow us to simply isolate propyl gallate and
to recycle the biocatalyst from the organic media. All of the above
are required for industrial production of propyl gallate.
3.4. Effect of surfactant on the activity of imprinted tannase
The effect of surfactant on the AF of tannase is shown in Fig. 5.
In Fig. 5a, five nonionic surfactants have a bigger effect on CR than
the ionic surfactant sodium lauryl sulfate (SLS). In particular, Tri-
ton X-100, as the optimal nonionic surfactant, makes an interfacial
imprinted tannase with a CR of approximately 18% and an AF of
1.8. Fig. 5b shows that the AF rises with increasing Triton X-100
concentration below 0.2%, at which the AF reaches its maximum.
It can be seen from Fig. 5 that nonionic surfactants have a posi-
tive effect on the activation of tannase, and just a small quantity
of surfactant, generally less than the critical micelle concentra-
tion (CMC) [21,22], can activate the enzyme. On the contrary, too
much surfactant reduces the catalytic activity of the enzyme, likely
by irreversible covalent inhibition [22]. Therefore, the appropriate
amount of surfactant is beneficial to the activation of the enzyme.
Previous studies [4,21,22,30] have reported that a few surfactants
can improve the catalytic performance of enzymes by enhancing
the rigidity of protein dynamics and substrate accessibility to the
active site and/or by inducing a more competent catalytic center in
anhydrous media [21]. In addition, surfactant can also enhance the
catalytic activity of the enzyme by diminishing the aggregation of
imprinted enzymes in anhydrous media.
A demonstration test was designed to investigate the role of
Triton X-100 in this work. Table 4 shows that the CR of the Triton
X-100-imprinted tannase (SE) is higher than that of the control (the
non-imprinted tannase with the surfactant, Es), and Es also has a
slight advantage over Ew in CR. According to the difference in CR
between SE and Es, it is believed that Triton X-100 can activate tan-
nase by bio-imprinting due to its interaction with tannase, which
favors a molecular conformation of tannase that is more stable and
similar to that of a highly active enzyme (shown in step 2 in Fig. 3).
By comparing the CR of Es with that of Ew, it is inferred that Triton
X-100 slightly enhances substrate accessibility to the active site of
the enzyme.
4. Conclusions
3.5. Effect of imprinting order on the activity of imprinted tannase
In summary, bio-imprinting can improve the activity of tan-
nase remarkably. The modification of tannase by the combined
use of various bio-imprinting methods created a promising bio-
catalyst with a surprising 40% enhancement of CR, 100-fold that
of the original control. Furthermore, joint application of a simple
The effect of the imprinting order on the AF of tannase was
investigated by analyzing the following series: (1) first pH imprint-
ing, second substrate (TA) imprinting, third interfacial imprinting

G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 32–37
37
immobilization method with the transesterification reaction in
organic media diminishes the production costs of propyl gallate.
Therefore, the synthesis of propyl gallate coupled with the exploita-
tion of TA on a large scale is a promising approach. This work not
only presents an effective way to make high-value use of various
TA-rich agro-forestry residues, but also broadens the applications
of bio-imprinting.
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The authors gratefully acknowledge financial support granted
to this work by Knowledge Innovation Program of the Chi-
nese Academy of Sciences (KSCX2-YW-G-050) and the National
High Technology Research and Development Program of China
(SQ2008AA02Z4477854), and thank Prof. Huang Qing and Prof.
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