Synthesis of propyl gallate from tannic acid catalyzed by tannase from Aspergillus oryzae: Process optimization of transesterification in anhydrous media

Article abstract of DOI:10.1016/j.molcatb.2012.06.003

Improving the catalytic capability of enzyme is an important challenge of biocatalysis in organic medium. Optimization of organic reaction system and reaction mode can elevate the catalytic ability. In order to enhance the catalytic efficiency of tannase-catalyzed transesterification from tannic acid, process parameters of the reaction and reaction mode were optimized further to improve the conversion rate of substrate. The result showed that with hexane as solvent, a conversion rate of substrate, 75%, was achieved at 40 °C in 20 mL reaction mixture composed of 0.75% water and 7.5% n-propanol, and that semicontinuous catalysis was the most favorable for production of propyl gallate, its average production rate was 2.5-fold that of batch catalysis. By SCC, a noted computative conversion rate of approximate, 90%, was obtained. Thus, it is expected that this study may present an efficient and ecofriendly method for industrial production of PG.

Full text of DOI:10.1016/j.molcatb.2012.06.003

Journal of Molecular Catalysis B: Enzymatic 82 (2012) 102–108
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Synthesis of propyl gallate from tannic acid catalyzed by tannase from Aspergillus
oryzae: Process optimization of transesterification in anhydrous media
Guangjun Niea,b,c, Hui Liua, Zhen Chena, Peng Wanga, Genhai Zhaoa, Zhiming Zhenga,∗
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:
Improving the catalytic capability of enzyme is an important challenge of biocatalysis in organic medium.
Optimization of organic reaction system and reaction mode can elevate the catalytic ability. In order
to enhance the catalytic efficiency of tannase-catalyzed transesterification from tannic acid, process
parameters of the reaction and reaction mode were optimized further to improve the conversion rate
of substrate. The result showed that with hexane as solvent, a conversion rate of substrate, 75%, was
achieved at 40 ◦C in 20 mL reaction mixture composed of 0.75% water and 7.5% n-propanol, and that
semicontinuous catalysis was the most favorable for production of propyl gallate, its average production
rate was 2.5-fold that of batch catalysis. By SCC, a noted computative conversion rate of approximate,
90%, was obtained. Thus, it is expected that this study may present an efficient and ecofriendly method
for industrial production of PG.
Received 16 March 2012
Received in revised form 15 May 2012
Accepted 4 June 2012
Available online 9 June 2012
Keywords:
Process
Propyl gallate
Transesterification
Tannase
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
gel [8], and adsorption [9] have been mainly used to improve the
efficiency. Otherwise, pH tunning [9] and optimization of reaction
An environmental-friendly way for chemicals synthesis has
such advantages as low energy consumption and no environmen-
tal contamination relative to a conventional chemical synthetic
medium [10–12] were also applied to better the efficiency.
Propyl gallate (PG) with excellent antioxidative capacity is usu-
ally used as food antioxidant [13]. It as a synthetic antioxidant is
very effective in improving the stability of biodiesel toward oxida-
tion [14] and in raising the oxidation onset temperature (OOT) [15].
In addition, PG also has potential pharmaceutical properties, such
as an antitumoral effect and an antinociceptive activity [16–20].
PG, in general, can be synthesized by a traditional chemical
catalysis means and an ecofriendly biocatalysis approach (shown in
Fig. 1). The chemical means tends to be replaced by the biocatalysis
approach owing to its high running temperature (80 ◦C) and high
corrosive environment caused by chemical catalyst (concentrated
sulfuric acid). As for biocatalytic synthesis of PG, it has two routes
including (1) two-step catalysis: TA → Gallic acid (GA) → PG; (2)
direct synthesis: TA → PG. The direct synthesis will become a hot
research topic due to its notable advantages, such as few steps and
lower reaction temperature (40 ◦C).
method. Biocatalysis will be
a main environmental-friendly
approach for chemicals manufacturing. Presently, to improve the
catalytic capability of enzyme is one of important challenges in bio-
catalysis with organic solvent as reaction medium. The catalytic
ability can be elevated by optimization of organic reaction system
and reaction mode. In the case of bio-synthesis of propyl gallate
(PG) by tannase-catalyzed transesterification, its conversional rate
of substrate (CR) is undesirable for commercial application till now.
Therefore, it is essential to enhance CR by process optimization of
transesterification in anhydrous media.
Tannase is a tannin acyl hydrolase (EC, 3.1.1.20) [1], which
can hydrolyze the ester and depside linkages in hydrolysable
tannins [2,3] and catalyze esterification between gallic acid and
alcohol [4] as well as the transesterification reaction between
tannic acid (TA) and gallic acid esters (e.g., PG) [5] (shown in
Fig. 1). In order to enhance the catalytic efficiency of tannase,
a few modifications have been reported. For example, a variety
of immobilization approaches, such as microencapsulation [6],
crosslinking-entrapment-crosslinking [7], microemulsion-based
However, a lower CR in the transesterification has been reported
in most of previous studies. For example, Gaathon et al. obtained
a CR of 5.1% by modulation of a reverse micelle system [10], and
Fernandez-Lorente et al. achieved a CR of 45% by optimizing the
diphase system consisted of water and 1-propanol [11].
In order to improve the CR, a tannase biocatalyst with high
transesterification-catalyzing performance was developed based
on bio-imprinting in our preliminary works, and it yielded a CR
of 40% without any process optimizations [21]. Nevertheless, the
∗
Corresponding author. Tel.: +86 551 5593148; fax: +86 551 5593148.
E-mail address: zhengzhiming2011@gmail.com (Z. Zheng).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.06.003

G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 102–108
103
Fig. 1. Different synthetic routes of propyl gallate from tannic acid include a two-step catalysis means and a direct catalysis approach. (1) TA is hydrolyzed into GA by microbial
tannase. (2) Microbial tannase catalyzes the synthesis of PG from GA by esterification. (3) Microbial tannase catalyzes the synthesis of PG from TA by transesterification. (4)
PG is synthesized by esterification from GA with concentrated sulfuric acid as a catalyst. The two-step catalysis contains: (1) step 1 + step 2; (2) step 1 + step 4. The direct
catalysis refers to the step 3 (transesterification catalyzed by microbial tannase or concentrated sulfuric acid).
biocatalyst is still a protein significantly affected by organic sol-
vent and reaction temperature. On the other hand, the inhibitions
resulted from substrate and product can weaken catalytic efficiency
of the modified enzyme. Therefore, the further improvement of the
CR in direct synthesis of PG must be subjected to the optimizations
of reaction system, temperature, and reaction mode.
As far as reaction mode is concerned, continuous catalysis (CC)
does not deactivate or consume the catalyst and further minimizes
or eliminates the need for multitudinous downstream separation
and purification steps, and thus it has been widely used in industrial
production [22].
enzyme-substrate solution was maintained without agitation for
5 min at ambient temperature.
Cryogenic protection (prior to lyophilization) and immobiliza-
tion: 12.5 ␮L 200 mM magnesium ions solution, 0.625 mL 1 M
mannose, 0.15 mL 10 mM Triton-X-100, and 0.5 g celite were put
in to the above solution, in turn, and then the mixture was agitated
for 5 min.
Lyophilization: The modified enzyme mixture was frozen at
−20 ◦C overnight, followed by lyophilization using a freeze dryer
(VirTis, SP Scientific USA). The lyophilized powders were stored at
4 ◦C until use.
In this paper, a modified tannase from Aspergillus oryzae was
utilized to catalyze the direct synthesis of PG from TA in anhydrous
media. The aim of this work is to further improve the CR of the
transesterification by optimization of catalysis system (i.e., solvent
type, solvent volume, water content, and n-propanol content), and
by investigation of the effects of run temperature, substrate, as well
as product on CR. In addition, three different reaction modes of
batch catalysis (BC), batch catalysis coupled with product separa-
tion (BCCPS), and semicontinuous catalysis (SCC) were analyzed. It
is attempted to construct an efficient and environmental-friendly
way to produce PG in an industrial scale.
2.3. Reaction mode
BC: An aliquot of modified tannase (containing 50 mg TA) was
added to approximate 20 mL reaction system composed of 1.5 mL
n-propanol, 18.5 mL hexane, and 0.15 mL distilled water. The trans-
esterification reaction was carried out at 40 ◦C and 200 rpm.
BCCPS: An aliquot of modified tannase (containing 50 mg TA)
was added to approximate 20 mL reactive system composed of
1.5 mL n-propanol, 18.5 mL hexane, and 0.15 mL distilled water. All
of the old reaction system including bulk product was replaced by
the equivalent fresh reaction system without TA every 12 h. The
transesterification reaction was carried out at 40 ◦C and 200 rpm.
SCC: An aliquot of modified tannase (containing 50 mg TA) was
added to approximate20 mL reaction system composed of 1.5 mL n-
propanol, 18.5 mL hexane, and 0.15 mL distilled water. All of the old
reaction system including bulk product was replaced by the equiv-
alent fresh reaction system containing 20 mg TA supplement every
24 h. The transesterification reaction was carried out in a 50 mL
conical flask at 40 ◦C and 200 rpm.
2. Experimental
2.1. Materials
Tannase from A. oryzae and PG (HPLC grade) were purchased
from Jinan Huazuan Trading Co., Ltd., China and from Sigma Co., USA
respectively. The other reagents were obtained from Sinopharm
Chemical Reagent Co., Ltd. (SCRC), China. The reagents are of an
analytical grade.
In these catalytic reactions, CR was defined as the mole per-
cent of TA completely transformed into PG relative to the whole TA
supplementation.
2.2. Preparation of a modified tannase
A modified tannase can be prepared according to the method
that is adapted based on our previous works [21].
2.4. Assay of propyl gallate
Modification (in aqueous media): 50 mg TA were dissolved
in the tannase solution, which included 16 IU tannase with
5 mL 90 mM pH 6.0 citrate buffer as solvent. The uniform
Samples were assayed by HPLC (Waters 600, Waters, USA)
equipped with Phenomenex C18 column with 250 × 4.60 mm, 4 ␮m

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G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 102–108
Fig. 2. Effects of solvent type, water content, and n-propanol content on the CR. (a) Effect of solvent type on the CR, water content, n-propanol content, and solvent volume
were designed as 1%, 10%, and 10 mL respectively; (b) effect of water content on the CR, both n-propanol content and solvent volume were the same as those mentioned in
Fig. 2a. Where, symbol square and triangle refer to hexane and petrol ether 30–60, respectively; (c) effect of n-propanol content on the CR, hexane was used as the solvent,
and water content was 1.5%. In all cases, the BC was chosen as the reaction mode, and the run time was 24 h. All of other conditions were the same as those mentioned in
Section 2.
at 35 ◦C. The mobile phase was composed of 50% methanol, 50%
pure water, and 0.01% acetate acid. 0.02 mL sample was detected
at 274 nm with standard PG as the control at a flow rate of 1 mL/min.
All of the reagents are of HPLC grade. All experiments were carried
out in duplicates unless stated otherwise.
From the above rule, it was found that determination of solvent
type also depends on the polarities of substrate and product. In
this work, hexane is a hydrophobic solvent that not only maintains
higher effective concentration of hydrophilic substrate, but also
removes the feedback inhibition of hydrophobic PG well. Further-
more, hexane has been well used in oil ester separation, biodiesel
production and pharmaceuticals [25–27] on account of its lower
toxicity and lower boiling point.
3. Results and discussion
3.1. Effect of solvent type on the conversion rate of substrate
3.2. Effect of water content and n-propanol content on the
conversion rate of substrate
Various organic solvents with different log P were screened for
their suitability in the synthesis of PG. The CRs with benzene (log
P = 2.0) and n-propanol (log P = 0.28) as solvents were evidently less
than those achieved with the four others as solvents (shown in
Fig. 2a) suggesting that the CR rises with the polarity of reaction
solvent decreasing. The maximum CR of 49% was achieved when
hexane (log P = 3.5) was used as solvent, followed by a CR of 45%
with petrol ether (30–60) as solvent.
As expected, non-polar solvents are superior for transesteri-
fication or esterification compared to hydrolysis since non-polar
solvents, such as hexane (log P = 3.5), cannot strip off the essential
water from a micro aqueous layers around an enzyme such that
the active conformation of the enzyme is retained [23]. The sol-
vent type in catalytic system can be determined according to the
following rules: |log Pi–log Ps| and |log Pcph–log Pp| should be min-
imal, but |log Pcph–log Ps| and |log Pi–log Pp| should be maximal
[24]. Where, log Pi, log Pcph, log Ps, and log Pp refer to the polari-
ties of the microenvironment of biocatalyst, the continuous organic
phase, the substrate, and the product, respectively.
The effect of water content between 0% and 3.0% in the reaction
system on the CR is shown in Fig. 2b. No matter in hexane or in
petrol ether (30–60), the CR rises with the water content increas-
ing up to 1.5%, while the content continues to increase it glides
slowly. It can be seen that water content in catalytic system dra-
matically affects the catalytic activity of enzyme as an appropriate
water content (generally 1–5% [9,28–30]) in the reaction system
can form the hydration shell around enzyme molecule that makes
the enzyme structure more desirable in the stability, flexibility and
catalytic performance of enzyme [31]. But, the shell is incompletely
formed at lower water content. On the other hand, the hyper activ-
ity of the modified enzyme disappears at higher water content. This
may be due to this fact that excessive water causes the enzyme
so more flexible in its conformation that the conformation with
hyper activity is changed. In addition, the requirement of water
activity is usually different for various types of catalytic reactions
catalyzed by the same enzyme. As for tannase, high water activity

G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 102–108
105
Fig. 3. Effect of solvent volume on the transesterification reaction. (a) Effect of solvent volume (5–30 mL) on the CR, water volume and n-propanol volume were designed as
150 ␮L and 1.5 mL respectively, and the run time was 24 h. (b) Effects of solvent volume (10 mL, 20 mL, and 30 mL) with time on the CR, both water volume and n-propanol
volume were same as those in Fig. 3a. Where, symbol square, circle, triangle, five-pointed star, and pentagon signify reaction time 3 h, 6 h, 9 h, 12 h, and 72 h, respectively;
(c) Effect of solvent volume (10 mL, 20 mL, and 30 mL) with time on the initial reaction rate, both water volume and n-propanol volume were same as those in Fig. 3a. In all
cases, the BC was chosen as the mode of catalysis reaction, and the run time was 24 h. All of other conditions were the same as those mentioned in Section 2.
will favor hydrolysis, whereas lower water activity will be better
for esterification or transesterification [32–34].
The dependence of the CR on n-propanol content between 5%
and 30% (v/v) was investigated. Fig. 2c indicates that the CR grows
with n-propanol rising up to 15%, at which the maximal CR, 50%,
was gained, and afterwards it diminishes. The explanation may
be that the increment of n-propanol (as a substrate) amount can
increase the effective concentration of the substrate at a lower alco-
hol concentration between 5% and 15%, while at a higher alcohol
concentration above 15%, the n-propanol (as a solvent) becomes an
toxic solvent disrupting hydration membrane of enzyme so that the
enzyme is inactivated [12]. Therefore, it is great important to make
n-propanol in the reaction mixture located in an optimal equilib-
rium between as a substrate and as a solvent. The same observation
has been presented by Yu et al. [12], but Sharma et al. have given
completely opposite findings [9].
Fig. 4. Effect of reaction temperature on the conversion rate (a) and the initial reac-
tion rate (b) in the transesterification reaction. The transesterification reaction was
carried out at various temperature and 200 rpm. Reaction system was the same as
3.3. Effect of solvent volume on the transesterification reaction
Effect of solvent volume between 5 mL and 30 mL on the CR is
shown in Fig. 3. As shown in Fig. 3a, the CR grows with increasing of
solvent volume up to 10 mL, afterwards, the CR enters into a plat-
form. Fig. 3b indicates the CRs in 10 mL, 20 mL, and 30 mL reaction
systems seem undifferentiated at the initial stage of the reaction,
however, the CR obtained in 20 mL reaction system is higher than
them achieved in the other two volumes of reaction system when
the reaction lasts for 72 h. The finding is agreement with the dif-
ference in the initial reaction rate (shown in Fig. 3c). It suggests
that excessive solvent can decrease the effective concentration of
substrate resulting in substrate deficiency, but a limited volume
of solvent can lead to the accumulation of the product so that the
progress of transesterification reaction is impeded at the ending
stage of the reaction.
that in the BC mentioned in the materials and methods. The PG yield was assayed
every 3 h, and the plot of PG yield vs. reaction time was fitted by polynomial model.
The apparent initial reaction rate can be calculated by derivation of the plot at 0 h.
Symbol square, circle, five-pointed star, triangle, and pentagon signify reaction time
3 h, 6 h, 12 h, 24 h, and 48 h, respectively.
temperature for the transesterification throughout 48 h, and that
the CRs gained at 45 ◦C and at 50 ◦C are higher than them achieved
at 35 ◦C and at 30 ◦C when the reaction are only carried out for 3 h,
whereas the CRs at 45 ◦C and at 50 ◦C are less than them at 35 ◦C
and at 30 ◦C when the reaction time extends up to 48 h. Fig. 4b also
indicates that 40 ◦C is the best temperature for the initial reaction
rate, and that both of the initial reaction rates at 45 ◦C and at 50 ◦C
exceed them at 35 ◦C and at 30 ◦C, respectively. It suggests that the
initial reaction rates are faster, but the average reaction rates in 48 h
at the higher temperatures are lower relative to them at the lower
ones. It may be due to this fact that the tannase is inactivated more
quickly over time at the higher temperature [35] so that its reac-
tion rate declines faster with time compared to that at the lower
3.4. Effect of temperature on the transesterification reaction
Effect of reaction temperatures between 30 ◦C and 55 ◦C on
the CR is shown in Fig. 4. Fig. 4a shows that 40 ◦C is the best

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G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 102–108
Fig. 5. Effects of substrate (a) and (b) and product (c) and (d) on the transesterification reaction. (a) Effect of TA addition (50–100 mg) with time on the yield of PG; (b) Effect
of TA addition (50–100 mg) on the initial reaction rate; (c) Effect of PG addition (0–50 mg) with time on the yield of PG; (d) Effect of PG addition (0–50 mg) on the initial
reaction rate. (a) and (b) An aliquot of modified tannase (containing 50 mg TA) and various supplemented TA were added to the reaction system. (c) and (d) An aliquot of
modified tannase (containing 50 mg TA) and various supplemented PG were added to the reaction system. All of the transesterification reactions were carried out at 40 ◦
C
and 200 rpm. The reaction system was the same as that in the BC mentioned in the materials and methods. The PG yield was assayed every 3 h, and the plot of PG yield vs.
reaction time was fitted by polynomial model. The apparent initial reaction rate can be calculated by derivation of the plot at 0 h. Symbol square, circle, triangle, five-pointed
star, and pentagon signify reaction time 6 h, 12 h, 24 h, 36 h, and 48 h, respectively.
temperature. The observation is similar to the findings reported by
Åkerman et al. [36]. Therefore, determination of a favorable reac-
tion temperature should be coordinated with such key parameters
as the initial reaction rate and the CR. Many previous studies have
given the same optimal reaction temperature, 40 ◦C [12,37]. But,
the optimal reaction temperature may be affected by the reaction
medium. For example, the reaction temperature decreased to ambi-
ent temperature when the bulk reaction media was composed of
water and n-propanol [11]. Accordingly, it is essential for the same
enzyme to investigate its optimal reaction temperature in various
reaction media.
more rapid with the reaction time extending. Fig. 5d indicates that
the initial reaction rate is also negative correlation with the incre-
ment of PG dose. It is demonstrated that the product supplement
inhibits the transesterification significantly by making the reaction
located in a new equilibrium with a lower CR earlier. Thus, it is great
crucial to relieve the feedback inhibition of product in commercial
production of PG.
Considering the effects of solvent volume, substrate, and prod-
uct on CR, selection of a favorable reaction mode is of remarkable
importance for further boosting increment of CR. It can evidently
remove the inhibitions resulted from unsuitable dose of substrate
and product.
3.5. Effect of substrate on the transesterification reaction
3.7. Effect of reaction mode on the transesterification reaction
Effect of TA dose between 50 mg and 100 mg on PG yield over
time is shown in Fig. 5a. The PG yields decline with increments of
TA dose before 12 h, but the yields increase with TA dose increas-
ing up to 70 mg when the reaction time extends, Fig. 5b also shows
that the initial reaction rate decreases with growth of TA dose. It
is referred that excessive TA can inhibit the catalytic activity of
tannase possibly by the formation of substrate–enzyme–substrate
complex [37], but the average reaction rate at a low amount of TA
reduces owing to the insufficient of substrate at ending stage of
the reaction. On the other hand, excessive TA may inhibit mass
transfer of substrate and of product due to the agglomeration
of hydrophilic TA in anhydrous media. Therefore, determination
of an appropriate dose of substrate is essential for keeping the
equilibrium between initial reaction rate and average reaction
rate.
Three reaction modes of BC, BCCPS, and SCC were studied fur-
ther to improve the CR and the productivity of PG, respectively.
Fig. 6a shows that the maximum CR obtained in the mode of BCCPS
in 96 h is 75%, it is evidently higher than previous reports [9–12,38].
Astonishingly, SCC is the better in PG yield than BCCPS (shown
in Fig. 6b), and its three parameters (i.e., PG yield, average pro-
duction rate, and catalytic efficiency of the tannase) are 2.5-fold
them achieved in BC in the same time (shown in Table 1). From
the slope of the profile between 24 h and 96 h in Fig. 6b, the CR
Table 1
Comparison of different reaction modes in PG production and catalytic efficiency of
the tannase.
Reaction mode
PG yield (SD)/␮mol
Average production
rate (SD)/␮mol/h
3.6. Effect of product on the transesterification reaction
BC
BCCPS
SCC
142.68 1.27
220.48 0.58
352.28 20.33
1.49 0.013
2.29 0.006
3.67 0.212
Effect of PG dose between 0 mg and 50 mg on PG yield with time
was studied. Fig. 5c shows that the PG yield declines with the incre-
ment of PG dose at 6 h, and the lowering rate becomes more and
Average production rate refers to the rate of the PG yield divided by the reaction
time (96 h).

G. Nie et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 102–108
107
Fig. 6. Effect of different catalysis modes on the conversion rate (a) and propyl gallate yield (b) the transesterification reaction. (a) Effect of different catalysis modes with
time on the CR; (b) effect of different catalysis modes with time on the initial reaction rate. Symbol square, circle, and triangle signify reaction mode BC, BCCPS, and SCC,
respectively. Reaction conditions were the same as those mentioned in Section 2.
of SCC will increase to around 61% if not considering the inacti-
vation of enzyme and the reaction lasting for a long time after
96 h. Based on these assumptions, the CR in the whole SCC reac-
tion is reckoned as about 90% (61% + (100%−61%) × 75%) provided
that substrate is no longer supplied to the reaction mixture after
96 h. Moreover, SCC has an overwhelming superiority not only in
the CR but also in the productivity of PG compared to BC and BCCPS
as supplement of small quantity of substrate coupled with prod-
uct separation by stages in SCC may be better in removing the
inhibitions resulted from substrate and product, and in improving
operating continuity and utilization rate of equipment and instal-
lations.
The relationship between PG yield and reaction time
(y = 0.000648x3−0.131x2 + 8.140x (Eq. (1), R2 = 0.993)) in BC
in 96 h is fitted by polynomial fitting to search the optimal time
interval for substrate supplement and product separation during
SCC. It is found from dy/dx = 0 based on Eq. (1) that the theoretical
maximum of PG yield (151.94 ␮mol) will appear when the reaction
lasts for 67.4 h. Nonetheless, the reaction time is too long to be
suitable for industrial production of PG. The calculation from Eq.
(1) finds the PG yield in 24 h reaches 128.86 ␮mol, 84.8% of that in
67.4 h. On the other hand, it can be seen from comparing BC with
BCCPS in Fig. 6 that the product inhibition before 24 h has a scarce
effect on the reaction rate. Thus, it manifests that the reaction
rate of BC is relatively satisfactory before 24 h. In view of the
convenience for practical operation in real industrial production,
the optimal interval of supplement and separation is decided as
24 h.
4. Conclusions
It is worth noting that the promising CR, 75%, was achieved
under the optimal conditions. The study of reaction mode found
that the SCC is the most favorable for PG production by transester-
ification, where a notable computative CR, approximate 90%, was
gained. Consequently, it is sure that this work, in principal, has con-
quered the block, which tannase activity is so low in catalyzing the
transesterification from TA to PG. The study of reaction mode also
reveals that continual production of PG by the biocatalytic method
is feasible in commercial scale. On the other hand, TA is found in a
variety of agro-forestry residues, such as wood, bark, leaves, fruits,
and galls [39]. Bearing in mind the natural raw material, high per-
formance biocatalyst, and preferable reaction mode, it is expected
that the biocatalytic synthesis of PG direct from TA exhibits enor-
mous economic and ecologic values, and thus it will be utilized to
commercially produce PG in environmental-friendly way.
Acknowledgements
This work has been supported by Knowledge Innovation Pro-
gram of the Chinese Academy of Sciences (KSCX2-YW-G-050) and
the National High Technology Research and Development Program
of China (SQ2008AA02Z4477854).
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A desired supplement of substrate should be determined to
relieve the inhibition of substrate better. As shown in Fig. 6a, 24 mg
(50 × 48%) of substrate is completely transformed into PG in 24 h in
BCCPS. Considering enzyme inactivation during reaction, a slightly
lower supplement of 20 mg TA is chosen for the further works.
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