Enhancement of propyl gallate yield in nonaqueous medium using novel cell-associated tannase of bacillus massiliensis

Article abstract of DOI:10.1080/10826068.2012.745873

Enzymatic synthesis of propyl gallate in organic solvent was studied using cell-associated tannase (EC 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was used as the biocatalyst. The effects of solvent, surfactant treatment, and bioimprinting on the propyl gallate synthesis were studied and subsequently optimized. Among various solvents, benzene followed by hexane was found to be the most favorable. Treatment of the biocatalyst with Triton X-100 at a lower concentration (0.2% w/v), before lyophilization, increased the propyl gallate yield by 24.5% compared to the untreated biocatalyst. The biocatalyst was imprinted with various concentrations of gallic acid and tannic acid. Biocatalyst imprinted with tannic acid showed 50% enhancement in the propyl gallate yield compared to the non-imprinted biocatalyst.

Full text of DOI:10.1080/10826068.2012.745873

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ENHANCEMENT OF PROPYL GALLATE
YIELD IN NONAQUEOUS MEDIUM USING
NOVEL CELL-ASSOCIATED TANNASE OF
Bacillus massiliensis
a
a
Mahesh Aithal & Prasanna D. Belur
a
Department of Chemical Engineering , National Institute of
Technology Karnataka , Surathkal , Srinivasnagar , India
Accepted author version posted online: 12 Dec 2012.Published
online: 14 Apr 2013.
To cite this article: Mahesh Aithal & Prasanna D. Belur (2013) ENHANCEMENT OF PROPYL GALLATE
YIELD IN NONAQUEOUS MEDIUM USING NOVEL CELL-ASSOCIATED TANNASE OF Bacillus massiliensis ,
Preparative Biochemistry and Biotechnology, 43:5, 445-455, DOI: 10.1080/10826068.2012.745873
To link to this article: http://dx.doi.org/10.1080/10826068.2012.745873
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Preparative Biochemistry & Biotechnology, 43:445–455, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6068 print/1532-2297 online
DOI: 10.1080/10826068.2012.745873
ENHANCEMENT OF PROPYL GALLATE YIELD IN NONAQUEOUS
MEDIUM USING NOVEL CELL-ASSOCIATED TANNASE OF
Bacillus massiliensis
Mahesh Aithal and Prasanna D. Belur
Department of Chemical Engineering, National Institute of Technology Karnataka,
Surathkal, Srinivasnagar, India
&
Enzymatic synthesis of propyl gallate in organic solvent was studied using cell-associated tan-
nase (EC 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was
used as the biocatalyst. The effects of solvent, surfactant treatment, and bioimprinting on the pro-
pyl gallate synthesis were studied and subsequently optimized. Among various solvents, benzene
followed by hexane was found to be the most favorable. Treatment of the biocatalyst with Triton
X-100 at a lower concentration (0.2% w=v), before lyophilization, increased the propyl gallate yield
by 24.5% compared to the untreated biocatalyst. The biocatalyst was imprinted with various
concentrations of gallic acid and tannic acid. Biocatalyst imprinted with tannic acid showed
50% enhancement in the propyl gallate yield compared to the non-imprinted biocatalyst.
Keywords bioimprinting, cell-associated tannase, gallic acid, lyoprotectants, nonaqu-
eous medium, propyl gallate, tannic acid
INTRODUCTION
Tannin acyl hydrolases have been known to carry out esterification or
transesterification reactions in nonaqueous media when gallic acid or tannic
acid is one of the substrate in producing gallic acid esters. Propyl gallate is
one such ester that has gained importance due to its widespread applications.
Current research is focused on the application of green chemistry approaches
to synthesize propyl gallate by using tannases as an alternative route to the con-
ventional chemical synthesis, which is found to be both costly and non-eco-
friendly. Current production methods developed by employing tannases as
alternative catalysts, although proving effective, need to be more economical,
faster, and more efficient to match the industrial requirements and replace the
Address correspondence to Prasanna D. Belur, Assistant Professor, Department of Chemical
Engineering. National Institute of Technology Karnataka, Surathkal, Srinivasnagar, Karnataka, India
575 025. E-mail: prsnbhat@gmail.com

446
M. Aithal and P. D. Belur
conventional methods. In our previous paper, we had described an application
of one such tannase, cell-associated tannase (CAT), to produce propyl gallate
from gallic acid and 1-propanol. The process was optimized and the effect of
[1]
each parameter was discussed. However, it was necessary to study various
factors that eventually help in enhancing the propyl gallate productivity.
Bioimprinting or molecular imprinting has been found to be useful in
[2]
achieving a better yield in the case of hydrolytic enzymes.
Molecular
imprinting locks the enzyme into a conformation that is favorable for catalysis
during lyophilization, through the addition of the desired substrate or a sub-
strate analog to the enzyme solution prior to freezing. Furthermore, the
ligand may prevent the formation of inactive ‘‘microconformations’’ in the
[3]
active site created during the lyophilization process. Enzyme inhibitors, sub-
[4]
[5]
strate analogs, and nucleophilic substrates have all been successfully used
as molecular imprints and have been shown to increase enzyme activity and
control enzyme selectivity (both enantioselectivity and substrate selectivity)
[6]
in organic media. Nie et al. attempted this approach in the case of tannases
with tannic acid as bioimprinting molecule along with surfactants and found
encouraging results. Apart from this, there are no reports available on bioim-
printed tannases, neither with gallic acid nor with tannic acid as substrate.
Compared to extracellular enzymes, cell-bound enzymes suffer low
activity, thereby giving low yield. Thus, efforts are required to expose them
more to substrates. Treatment with surfactants (mainly nonionic surfac-
tants) is an option to selectively permeabilize cells to expose more
cell-bound enzymes. The concept of surfactant coating is not new and has
[
7]
been successfully employed in the case of extracellular lipases
and
[6]
tannases.
This technique has also been found to improve surfactant
[
8]
coated lipase expression on yeast when used at appropriate concentra-
tions, and has been shown to increase cell hydrophobicity, enabling the
use of more hydrophobic solvents as the reaction media.
In the current paper, we discuss production improvement strategies for
a unique naturally immobilized tannase. The biomass of Bacillus massiliensis
showing CAT activity was used to carry out propyl gallate synthesis in
nonaqueous media. The effect of solvents and surfactant treatment was
initially studied and optimized. Later, use of tannic acid as a substrate
instead of gallic acid was explored. Finally, the effect of bioimprinting with
gallic acid and tannic acid on propyl gallate yield was studied.
MATERIALS AND METHODS
Microorganism and Maintenance
The bacterium Bacillus massiliensis exhibiting CAT, which was isolated
[1]
and identified earlier by Belur et al.,
was used for this study. The

Enhancement of Propyl Gallate Yield
447
bacterium was deposited in the ‘‘Microbial Type Culture Collection
and Gene Bank’’ (MTCC), Institute of Microbial Technology (IMTECH
Chandigarh, India), bearing the number MTCC 8930, and the Gene-Bank
accession number for the nucleotide sequence is AB633324. The culture
was maintained by monthly transfer on to nutrient agar slants and stored
ꢀ
ꢀ
at 5 C after incubating at 30 C for 24 hr.
Biomass Production, Harvesting, and Lyophilization
The inoculum was developed by adding a loop full of culture to 100 mL
ꢀ
nutrient broth and incubating it for 12 hr at 30 C in an incubator shaker;
2
2
mL of this inoculum was added to 100 mL production medium in
50-mL Erlenmeyer flasks, having the composition (g=L): glucose, 5; tryp-
tose (HiMedia, India), 2; and gallic acid, 0.4, with initial pH 5. The culture
ꢀ
was incubated in a rotary shaker at 250 rpm for 25 hr at 30 C. The biomass
containing the tannase enzyme was separated from the fermentation broth
by centrifuging the suspension at 5500 ꢁ g for15 min, washing twice with cit-
ꢀ
rate buffer (0.1 M), pH 5.0, and storage at 5 C in the form of the pellet. The
required amount of biomass was taken and 1.5 mL of 0.1 M citrate buffer
ꢀ
ꢀ
(
pH 5.0) was added and frozen at ꢂ20 C and lyophilized at ꢂ40 C. The lyo-
philized powder was used as the biocatalyst for esterification. The enzyme
activity of lyophilized powder was measured according to the method
described by Belur et al. One unit of enzyme activity is defined as 1 mmol
mol gallic acid released per minute under the assay conditions. Only those
batches having enzyme activity of 4 ꢃ 0.2 U=L, corresponding to 0.9 U=g of
dry cell weight (DCW), was used for the experiments.
[
9]
Reaction Setup
All the reaction components, namely, biocatalyst, substrates, and solvent,
were equilibrated to water activity of 0.33 for 3 days prior to the reaction over
saturated salt solution of MgCl ꢄ 6H O. Solvents were dried over molecular
2
2
sieves prior to use. Reaction components were mixed in 15-mL Teflon lined
tubes and the reaction was carried out at 200 rpm in an orbital shaker (Scie-
ꢀ
genics India) at 30 C for 48 hr. Typical reaction mixture consisted of 40.4 mg
biocatalyst, 0.4 mM gallic acid, dissolved in 6.52% (v=v) 1-propanol in 9.5 mL
benzene. Control experiments were also carried out without the biocatalyst.
All the experiments were done in duplicate and the mean values were reported.
Propyl Gallate Estimation
The propyl gallate yield was analyzed using a method reported by Yu
[10]
et al. : 200 ml of biocatalyst free reaction mixture was taken and diluted

448
M. Aithal and P. D. Belur
with 2 mL 0.15 mM ethyl paraben (ethyl 4-hydroxy benzoate) in methanol.
Twenty microliters of this sample was injected into a Dionex HPLC system
(
5
Dionex, NJ) fitted with an Agilent Eclipse XDB-C18 column (4.6 mm ꢁ
mm ꢁ 15 cm). Mobile phase consisted of methanol:water (55:45), with
pH adjusted to 3 with o-phosphoric acid. The flow rate was set to 1 mL=
min. Detection was carried out at 275 nm using an ultraviolet (UV) detector
ꢀ
at 30 C. Ethyl paraben was the internal standard, and the yield was calcu-
lated based on the calibration curve of propyl gallate to ethyl paraben area
versus standard propyl gallate concentration (mg=mL) (y ¼ 0.0.0304x ꢂ
2
0
.0019; R ¼ 0.9717, where x is propyl gallate yield in mg=mL and y is the
ratio of propyl gallate peak area to ethyl paraben peak area).
Effect of Different Solvents
Benzene in the reaction mixture was replaced with other solvents with
different log P values. Acetone, hexane, diethyl ether, toluene, and hep-
tanes were the solvents chosen. To maintain consistent water content, all
these solvents were also equilibrated to water activity (a ) of 0.33.
w
Effect of Surfactant Treatment
Different surfactants, both ionic (sodium dodecyl sulfate [SDS]) and
nonionic (Triton X-100, Tween 80), were taken for the study. Different
concentrations of each surfactant were prepared in 0.1 M citrate buffer
of pH 5.0. Concentration ranges were 0.5, 1, 2, 5, and 10% (w=v) for SDS
and 0.5, 1, 2, 5, and 10% (v=v) for Triton X-100 and Tween 80.
CAT was prepared as explained earlier. Once the cell pellets were
washed, 10 mL of these surfactant solutions was added to the cell pellets
obtained from 100 mL broth (0.45 g DCW). These were then incubated
ꢀ
for 4 hr with intermittent mixing at 4 C. After incubation, surfactant was
washed repeatedly with buffer and centrifuging at 5500 ꢁ g to obtain
surfactant-free cell pellet. This pellet was suspended in 0.1 M citrate buffer
(
pH 5.0) and lyophilized.
Effect of Tannic Acid as Substrate
This experiment was conducted to check the ability of cell-associated
tannase to carry out its transesterification with tannic acid as the substrate.
The reaction mixture consisted of 40.4 mg biocatalyst powder (lyophilized
in the presence of buffer) and tannic acid (0.1, 0.25, 0.5, 1, 2, 5 and 10 mM)
dissolved in 6.52% (v=v) 1-propanol and 9.5 mL benzene. The reaction was

Enhancement of Propyl Gallate Yield
449
ꢀ
carried out for 48 hr at 30 C at 200 rpm in an orbital shaker after equili-
bration to a 0.33 for 3 days. Samples were analyzed after 48 hr.
w
Effect of Bioimprinting with Gallic Acid and Tannic Acid
The harvested biomass showing cell-associated tannase activity was added
with 1 mL of gallic acid of varying concentration (0.01, 0.025, 0.05, 0.1, 0.25,
0.5, and 1% w=v) dissolved in citrate buffer (0.1 M, pH 5.0) so as to achieve
gallic acid to DCW ratio of 0.25, 0.625, 1.25, 2.5, 6.25, and 12.5 mg=g. The
ꢀ
mixture was incubated for 2 hr with intermittent mixing at 4 C and lyophi-
lized. A control was also prepared without gallic acid treatment. Similarly,
biocatalyst was imprinted with tannic acid as well. Tannic acid to DCW ratio
of 1.25, 2.5, 6.25, 12.5, 25, 62.5, and 125 mg=g was prepared and incubated
ꢀ
for 2 hr at 4 C and lyophilized. The propyl gallate was produced by using this
lyophilized biomass having cell-associated tannase activity. The reaction mix-
ture consisted of 40.4 mg imprinted lyophilized powder, 2 mM tannic acid,
6.52% (v=v) 1-propanol, and 9.5 mL benzene. Reaction components were
equilibrated to a 0.33 for 3 days and the reaction was carried out for
w
ꢀ
4
8 hr at 30 C and at 200 rpm in an orbital shaker. A control experiment
was conducted using non-imprinted lyophilized biocatalyst.
RESULTS AND DISCUSSIONS
Effect of Different Solvents
It is clear from Figure 1 that benzene suited better than the other sol-
[10,11]
vents. This is consistent with the earlier reports.
Hexane gave a similar
FIGURE 1 Effect of different solvents on propyl gallate yield. Biocatalyst, 40.4mgþ 0.4 mM gallic acid
ꢀ
þ 6.52% (v=v) 1-propanol þ 9.5 mL solvent (all equilibrated to a
w
0.33) shaken at 200rpm for 48hr at 30 C.

450
M. Aithal and P. D. Belur
product yield as that of the benzene (19.75 mg=mL versus 20.16 mg=mL). In
nonaqueous enzymology it is customary to prefer solvents which are hydro-
phobic (log P > 2) in nature. Our results show that the increase in hydropho-
bicity did not give better yield beyond benzene. Thus, these results reiterate
that there is no relation between log P value of a solvent and the production
[
12]
of propyl gallate. Halling
observed that hydrophobicity merely may not
[13]
be enough to correlate with increase in yield. Secundo et al.
have postu-
lated that along with hydrophobicity, bulkiness also can influence enzyme
activity since some molecules of solvent may interact with the binding site
of enzymes to form solvent–enzyme complex. The complex would behave
differently depending on the nature of solvent. In case of hydrophilic
solvents, solvents may deactivate the enzyme in the way of disrupting the
functional structure of the enzyme or stripping off the essential water from
the enzyme. This is because water has higher affinity in hydrophilic solvent
rather than when bound to the enzyme. As a consequence, the catalytic
activity of the enzyme was decreased due to the lack of bound water to pre-
[14]
serve the enzyme conformation flexibility.
This structural mobility is
necessary for its catalytic action. There are also reports that the solvent with
a branched or a ring structure is more suitable for the enzymatic reaction
[15,16]
compared to the solvent with a straight chain.
Effect of Surfactant Treatment
Triton X-100 gave a better result at a concentration of 0.2% (w=v)
compared to the other surfactants (Figure 2). There was 24.5% increase
in propyl gallate yield from untreated control reaction, indicating that
the surfactant had exposed more enzyme active sites leading to enhanced
esterification activity. There was about 17.2% increase in yield when CAT
was treated with 0.1% Triton X-100, indicating that a slight change in the
surfactant concentration in 0.1–0.2% (w=v) range may not affect the yield
much. However, Triton X-100 had a negative effect on propyl gallate yield
beyond 2% (w=v). This was also the case in aqueous systems, wherein 0.2%
(
w=v) treated lyophilized biocatalyst showed marked increase in the activity
compared to the untreated biocatalyst (1.2 U=g versus 0.5 U=g, Table 1).
[17]
This is in agreement with the earlier reports on lipases.
The toxicity
of Triton X-100 molecules is due to the disrupting action of its polar head
group on the hydrogen bond present within the cell’s lipid bilayer, leading
to the destruction of the compactness and integrity of the lipid membrane.
The insertion of a detergent monomer into the lipid membrane begins at
low concentrations. This leads to disruption of the cellular structure and
eventually overpermeabilization of the cell membrane at concentrations
above the critical micelle concentration (CMC, 0.02–0.03% w=v) from the

Enhancement of Propyl Gallate Yield
451
FIGURE 2 Effect of surfactant treatment of whole cell biocatalyst on propyl gallate yield. Biocatalyst,
4
0.4 mg þ 0.4 mM gallic acid þ 6.52% (v=v) 1-propanol þ 9.5 mL benzene (all equilibrated to a
w
0.33)
ꢀ
shaken at 200 rpm for 48 hr at 30 C.
bilayer–micelle transition. In our case, the Triton X-100 concentration was
well above the CMC, but the enzyme activity was still intact, which was quite
remarkable.
In the case of Tween 80, concentration of 0.05% (w=v) showed only 4.4%
increase in propyl gallate yield, whereas higher concentrations led to drastic
drop in the yield. This was again confirmed by the enzyme activity in aque-
ous system (0.6743 U=g versus 0.5 U=g). This may be due to much harsher
interactions between Tween 80 and the cells compared to Triton X-100.
Much higher denaturation was observed with SDS, which resulted in a very
low enzyme activity (0.0025 U=g versus 0.5 U=g for 0.05%w=v treated bio-
catalyst; Table 1) and also propyl gallate yield in the nonaqueous system
(
5.08 mg=mL with 0.05% w=v SDS treated biocatalyst). The anionic detergent
SDS was harsher than the already mentioned nonionic detergents and
found to be detrimental even at lower concentrations. SDS is known to bind
ꢅ
TABLE 1 Effect of Surfactant Treatment on Tannase Activity of Biocatalyst
Enzyme Activity after Lyophilization (U=g)
Surfactant
Used (w=v)
0.05%
0.1%
0.2%
0.5%
1%
2%
Triton X-100
Tween 80
SDS
0.502
0.674
0.003
0.913
0.401
0.001
1.2
0.299
0.001
0.345
0.016
0
0.23
0
0
0
0
0
ꢅ
4
0.4 mg biocatalyst þ 0.4 mM gallic acid þ 6.52% (v=v) 1-propanol þ 9.5 mL
ꢀ
benzene (all equilibrated to a
w
0.33) shaken at 200 rpm for 48 hr at 30 C.

452
M. Aithal and P. D. Belur
cooperatively to most proteins, with the critical concentration for cooperat-
ive binding being about 25% of the critical micelle concentration (CMC).
Nonionic surfactants rarely induce cooperative binding and therefore do
not usually denature proteins, although dissolution into inactive or less
active subunits can occur. Therefore, anionic surfactants such as SDS prob-
ably interact at low concentrations with the cell wall proteins to produce a
complex. Such a complex, in the presence of excess surfactant, would
induce conformational changes or influence the active site of tannase and
produce inhibition. Such interactions between the charged head group of
the surfactants and oppositely charged enzyme have been found to affect
three-dimensional structures affecting the enzyme’s catalytic activity.
Conversely, for nonionic surfactants, such conformational changes would
hardly occur until high concentrations are reached because of weak electro-
static interactions of the surfactant with the enzyme and also with the active
[18]
site.
Effect of Tannic Acid as the Substrate
Gallic acid was replaced with different concentrations of tannic acid to
check the ability of the CAT to transesterify tannic acid in the presence of
1-propanol. It was found that CAT was having transesterification capacity in
addition to esterification at the same water activity. The tannic acid con-
centration was found to be affecting propyl gallate yield significantly. It
increased initially, showing a maximum value with 2 mM tannic acid and
decreased henceforth (Figure 3). On a molar basis, this corresponded to
FIGURE 3 Propyl gallate yield as a function of tannic acid concentration. Biocatalyst, 40.4 mg þ tannic
acid þ 6.52% (v=v) 1-propanol þ 9.5 mL benzene (all equilibrated to a
w
0.33) shaken at 200 rpm for
ꢀ
4
8 hr at 30 C.

Enhancement of Propyl Gallate Yield
453
10.4% yield, which is still less compared to the molar yield with gallic acid as
the substrate (25%). This is comparable with the earlier reports. Sharma
[
19]
and Gupta
used 10 mM tannic acid to obtain about 15% product yield.
[
6]
Nie et al. have reported a marked increase in propyl gallate yield while
using tannic acid as the substrate.
Interestingly, tannic acid is known to precipitate proteins forming aggre-
gates; hence, transesterification with tannic acid is usually not carried out
with free tannase but with immobilized tannase, as immobilization was found
to be not favoring this aggregation. In our case, tannase was already naturally
immobilized in the cell and no further immobilization was required. There
was no visible protein precipitation or macromolecular aggregations.
Effect of Bioimprinting on Propyl Gallate Yield
From Figure 4 it is evident that biocatalyst imprinted with 0.625 mg gallic
acid=g DCW gave the highest product yield compared to the others. There
was about 49.5% increase in the yield compared to the non-imprinted con-
trol (30.125 mg=mL versus 20.15 mg=mL). There was also an increase in the
yield initially, which then decreased gradually beyond 0.625 mg=g of gallic
acid. This may be due to the fact that gallic acid at higher concentrations
is inhibitory for esterification to occur. The imprinted gallic acid can also
redissolve into the solvent system during reaction, increasing the effective
gallic acid concentration beyond the inhibition level. At lower gallic acid
concentrations, although such a process may be possible, there are already
more enzyme active sites preserved during lyophilization to avoid substrate
inhibition. Considering that the solubility of gallic acid is low in most
FIGURE 4 Effect of gallic acid bioimprinting on propyl gallate yield. Biocatalyst, 40.4 mg þ 0.4 mM
gallic acid þ 6.52% (v=v) 1-propanol þ 9.5 mL benzene (all equilibrated to a
w
0.33) shaken at 200 rpm
ꢀ
for 48 hr at 30 C.

454
M. Aithal and P. D. Belur
FIGURE 5 Effect of bioimprinting with tannic acid on propyl gallate yield. Biocatalyst, 40.4 mg þ 2 mM
tannic acid þ 6.52% (v=v) 1-propanol þ 9.5 mL benzene (all equilibrated to a
w
0.33) shaken at 200 rpm
ꢀ
for 48 hr at 30 C.
organic solvents, and also the solvent toxicity on biocatalysts, gallic acid was
not washed off from the biocatalyst after imprinting, in order to check
whether this bound gallic acid could be released during reaction.
As evident from Figure 5, CAT imprinted with 12.5 mg of tannic acid=g
DCW showed higher yield. There was about 50% increase in the yield com-
pared to the non-imprinted enzyme. The yield decreased drastically at
[6]
higher concentrations of TA. Similar effects were observed by Nie et al.,
who reported a 4.86-fold increase in the conversion rate with a combined
strategy of pH, TA imprinting, and temperature.
Tannic acid is known to inhibit the biocatalytic activity of tannase by
changing the pH value in the hydrated shell around the enzymes. When
imprinted with tannic acid, at an appropriate pH tuned with pH imprinting,
tannase is believed to form an efficient and specialized active center, form-
ing transition complex of substrate-enzyme at the appropriate concen-
tration of TA. However, as the concentration of TA increases, free tannase
becomes very ‘‘sticky’’ due to molecular aggregation, which prevents
diffusion of the substrate and product into and out of the complex, respect-
[6]
ively, and thus the apparent activity is reduced. Perhaps due to the same
reason, the yield reduced when a higher concentration of TA was used for
imprinting in this study.
CONCLUSIONS
According to this study it can be concluded that hydrophilic and hydro-
phobic solvents were detrimental for propyl gallate synthesis. Treatment of

Enhancement of Propyl Gallate Yield
455
the biomass prior to lyophilization with Triton X-100 increased the product
yield considerably. Bioimprinting with gallic acid was also found to enhance
the yields, suggesting a reason for molecular imprinting gaining importance
in nonaqueous enzymology. CAT also showed considerable tranesterifica-
tion activity, which is an interesting prospect in producing propyl gallate
economically. A combined approach of surfactant treatment coupled with
bioimprinting with substrate enhanced the propyl gallate yield by 2.65 times.
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