Synthesis of propyl gallate by transesterification of tannic acid in aqueous media catalysed by immobilised derivatives of tannase from Lactobacillus plantarum

Article abstract of DOI:10.1016/j.foodchem.2011.02.057

Immobilised derivatives of tannase from Lactobacillus plantarum were able to catalyse the transesterification of tannic acid by using moderate concentrations of 1-propanol in aqueous media. Transesterification of tannic acid was very similar to transesterification of methyl gallate. The synthetic yield depended on the pH and concentration of 1-propanol, although it did not vary much when using 30% or 50% 1-propanol. Synthetic yields of 45% were obtained with 30% of 1-propanol at pH 5.0. The product was chromatographically pure, and the reaction by-product was 55% pure gallic acid. On the other hand, immobilised tannase was fairly stable under optimal reaction conditions.

Full text of DOI:10.1016/j.foodchem.2011.02.057

Food Chemistry 128 (2011) 214–217
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Short communication
Synthesis of propyl gallate by transesterification of tannic acid in aqueous
media catalysed by immobilised derivatives of tannase from Lactobacillus plantarum
Gloria Fernandez-Lorente a, Juan Manuel Bolivar b, Javier Rocha-Martin b, Jose A. Curiel a, Rosario Muñoz a,
Blanca de las Rivas a, Alfonso V. Carrascosa a, Jose M. Guisan b,
⇑
a Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
b Instituto de Catalisis, CSIC, Campus UAM-Cantoblanco, 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 April 2010
Received in revised form 16 January 2011
Accepted 14 February 2011
Available online 26 February 2011
Immobilised derivatives of tannase from Lactobacillus plantarum were able to catalyse the transesterifica-
tion of tannic acid by using moderate concentrations of 1-propanol in aqueous media. Transesterification
of tannic acid was very similar to transesterification of methyl gallate. The synthetic yield depended on
the pH and concentration of 1-propanol, although it did not vary much when using 30% or 50% 1-propa-
nol. Synthetic yields of 45% were obtained with 30% of 1-propanol at pH 5.0. The product was chromato-
graphically pure, and the reaction by-product was 55% pure gallic acid. On the other hand, immobilised
tannase was fairly stable under optimal reaction conditions.
Keywords:
Transesterifications in aqueous media
Natural antioxidants
1-Propanol
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
intermediary compound in the synthesis of the antibacterial drug,
trimethoprim, which is used in the pharmaceutical industry (Sittig,
Propyl gallate is a very interesting food antioxidant because it
prevents lipid oxidation (Adegoke et al., 1998). In addition, the fol-
lowing interesting health benefits have been reported: antitumoral
effects, prevention of mutagenesis, and scavenging of free radicals
(Chung, Wong, Wei, Huang, & Lin, 1998). Tannic acid is a vegetal
residue, and its direct enzymatic transformation into propyl gallate
converts the synthetic propyl gallate into a natural antioxidant
additive.
Tannases or tannin acyl hydrolases (EC, 3.1.1.20) catalyse the
hydrolysis reaction of the ester bonds present in the hydrolysable
tannins and gallic acid esters. Tannases are usually induced in
many microorganisms grown in the presence of tannic acid. Osa-
wa, Kuroiso, Goto, and Shimizu (2000) first reported tannase activ-
ity in Lactobacillus plantarum isolates. This was later confirmed in L.
plantarum strains isolated from various food substrates (Nishitani
& Osawa, 2003; Nishitani, Sasaki, Fujisawa, & Osawa, 2004; Va-
quero, Marcobal, & Muñoz, 2004). It has been postulated that this
enzymatic property has an ecological advantage for this species, as
it is often associated with fermentation of plant materials.
At the moment, the largest commercial applications of tannases
are the elaboration of instant tea and the production of gallic acid
(Chae & Yu, 1983; Coggon, Graham, & Sanderson, 1975; Pourrat,
Regerat, Pourrat, & Jean, 1985). Gallic acid is an important
1988); gallic acid is also very useful in the food industry because it
is a substrate for the chemical or enzymatic synthesis of propyl gal-
late, a potent antioxidant. Additionally, tannase is used as a clarify-
ing agent in some wines and fruit juices (Lekha, Ramakrishna, &
Lonsane, 1993).
Very interesting enzymatic biotransformations of tannic acid or
gallic acid into propylgallate in anhydrous media have already
been reported (Gaathon, Gross, & Rozhanski, 1989; Sharma & Gup-
ta, 2003; Yu & Li, 2006; Yu, Li, & Wu, 2004). Polar organic solvents
usually promote important inactivations of enzyme derivatives,
but very few of them are compatible with food technology (e.g.,
methyl ethyl ketone). The performance of propyl gallate (or other
esters) synthesis in aqueous media has rarely been reported, and
it could provide important technological advantages (Acosta, Filice,
Fernandez-Lorente, Palomo, & Guisan, 2011). The substrate is a
natural residue, the reaction media (mixtures of water and 1-pro-
panol) is innocuous and the catalyst is an immobilised GRAS
enzyme.
At first glance, 1-propanol is not a natural substrate of tannases;
hence, a correct adsorption of 1-propanol on the active centre of all
tannases should not be expected. However, tannases are very
abundant, and some of them could use 1-propanol as an enzyme
acceptor in aqueous media.
In previous papers, immobilisation–stabilisation of tannase
from L. plantarum has been reported (Curiel et al., 2010; Mateo
et al., 2010). With one of these derivatives, a successful and
⇑
Corresponding author.
E-mail address: jmguisan@icp.csic.es (J.M. Guisan).
0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.02.057

G. Fernandez-Lorente et al. / Food Chemistry 128 (2011) 214–217
215
complete hydrolysis of tannic acid to yield chromatographically
pure gallic acid has been reported (Curiel et al., 2010). In the pres-
ent communication, the transesterification catalysed by immobi-
lised tannase in aqueous media was studied. The direct
conversion of tannic acid into propyl gallate was intended. Optimal
synthetic conditions were designed. Under these conditions, the
stability of enzyme derivative and the purity of the reaction prod-
ucts were also analysed.
for 24 h during the immobilisation process. A reference suspension
of reduced glyoxyl-agarose, was used to discard unspecific adsorp-
tions. At the end-point of the reaction, sodium borohydride
(20 mg) was added, and the mixture was maintained at 20 °C with
gentle stirring. After 30 min, the immobilised derivatives were
washed thoroughly with distilled water (Guisán, 1988).
2.5. Inactivation of different immobilised derivatives of tannase in the
presence of water–organic solvent
2. Materials and methods
Enzyme derivatives were washed with an aqueous phase
achieved after equilibrating the solutions of the desired water/or-
ganic solvent mixture at pH 5, 25 °C, and 30% of n-propane. Subse-
quently, the enzyme derivatives were resuspended in this solution
and incubated at the temperature indicated. Samples were period-
ically withdrawn, and activity was assessed, following the assay
conditions described above. Experiments were carried out in trip-
licate, and the standard error was never over 2%.
2.1. Materials
Crosslinked 10% agarose was purchased from Agarose Bead
Technologies (Madrid, Spain). The L. plantarum CECT 748T strain
was purchased from the Spanish Culture Type Collection (CECT).
Escherichia coli DH5R was used for all DNA manipulations. E. coli
JM109 (DE3) plasmid was used for expression from the pURI3 vec-
tor (Pourrat et al., 1985). Methyl gallate and tannic acid were ob-
tained from Fluka. All other reagents were of analytical grade.
2.6. Enzymatic synthesis of propyl gallate from methyl gallate and
tannic acid
2.2. Enzymatic assay: hydrolysis of methyl gallate
One hundred milligrams of immobilised preparation were
added to 5 ml of substrate, 2 mM tannic acid or 10 mM methyl gal-
late in 50 mM ammonium acetate buffer at pH 5 and 25 °C under
continuous stirring. The transesterification reaction was carried
out in the presence of 30% 1-propanol. The product conversion
was analysed by RP-HPLC (Spectra Physic SP 100 coupled with a
UV detector Spectra Physic SP 8450), using a Kromasil C18
(25 cm  0.4 cm) column. Products were eluted at a flow rate of
1.0 ml/min, using methanol–10 mM sodium acetate at pH 2.95
(35:65, v/v), and UV detection was performed at 280 nm. Retention
times were as follows: 1.7 min for tannic acid, 3.4 min for methyl
gallate and 12.5 min for propyl gallate.
The esterase activity of tannase was determined, using a rhoda-
mine assay that was specific for gallic acid (Inoue & Hagerman,
1988). Rhodamine reacts only with gallic acid and not with any
other galloyl esters or other phenolics. Therefore, gallotannins
are measured by determining the quantity of gallic acid formed
after the hydrolysis of the tannins. Gallic acid analysis in the reac-
tions was determined using the following protocol: extract ali-
quots of 100
gallate in phosphate buffer (50 mM, pH 6.5) for 10 min at 37 °C.
After this incubation, 150 l of a methanolic rhodamine solution
(0.667% w/v rhodamine in 100% methanol) were added to the mix-
ture. After 5 min of incubation at 30 °C, 100 l of 500 mM KOH
were added, and the mixture was diluted to 900 l with distilled
ll were incubated with 100 ll of 25 mM methyl
l
l
l
2.7. Inhibition of the enzymatic hydrolysis of propyl gallate by gallic
acid and by 1-propanol
water. After an additional incubation of 5–10 min, absorbance at
520 nm was spectrophotometrically measured. A standard curve,
using gallic acid concentrations ranging from 0.125 to 1 mM, was
prepared. One unit of tannase activity was defined as the amount
One hundred milligrams of glyoxyl-tannase derivative were
added to 10 ml of 10 mM propyl gallate substrate in the presence
of 10 mM gallic acid and 30% 1-propanol in ammonium acetate
buffer at pH 5 and 25 °C under continuous stirring. The hydrolysis
reaction was carried out in a pH-stat Mettler Toledo DL50 graphic
in order to maintain the pH value constant during the reaction. The
conversion was analysed by RP-HPLC (Spectra Physic SP 100 cou-
pled with an UV detector Spectra Physic SP 8450) using a Kromasil
C18 (25 cm  0.4 cm) column. Products were eluted at a flow rate
of 1.0 ml/min, using methanol–10 mM sodium acetate at pH 2.95
(35:65, v/v), and UV detection was performed at 280 nm. Retention
times were as follows: 1.7 min for tannic acid, 3.4 min for methyl
gallate and 12.5 min for propyl gallate.
of enzyme required to release 1
lmol of gallic acid per minute un-
der standard reaction conditions.
2.3. Immobilisation on CNBr-activated agarose
Expression of recombinant tannase and the protocol for enzyme
purification have been previously described (Curiel et al., 2009).
Immobilisation of the enzyme on CNBr-activated support was per-
formed at pH 7 for 15 min at 4 °C to reduce the possibility of for-
mation of an intense multipoint covalent attachment (Mateo
et al., 2005). Thus, it may be assumed that immobilisation on this
support, under these conditions, will not alter the enzyme struc-
ture. Five grams of CNBr-activated support were added to a solu-
tion of 50 ml of purified tannase. After 15 min, approximately
30% of enzyme became immobilised on the support, and the activ-
ity was almost unaltered. The enzyme-support immobilisation was
finished by incubating the support with 1 M ethanolamine at pH 8
for 2 h. Finally, the immobilised preparation was washed with
water.
3. Results and discussion
3.1. Transesterification of methyl gallate and tannic acid in aqueous
media catalysed by immobilised tannase from L. plantarum (LPT)
LPT was able to catalyse the transesterification of methyl gallate
by 1-propanol. Initial ratios between propyl gallate and gallic acid
slightly depended on pH and the concentration of cosolvents (Ta-
ble 1). At first glance, pH 5.0 and 50% 1-propanol promotes the
highest synthetic yield (55%). However, the enzyme derivatives
are poorly stable in the presence of highly concentrated co-sol-
vents; hence, pH 5.0 and 30% of co-solvent were selected as suit-
able reaction conditions (synthetic yield of 45%).
2.4. Immobilisation of tannase on glyoxyl-agarose supports
One gram of glyoxyl-support (75 lmol/ml) was added to 40 ml
of enzyme solution containing 0.8 mg of pure tannase in bicarbon-
ate buffer (100 mM, pH 10.0). The mixture was maintained at 25 °C

216
G. Fernandez-Lorente et al. / Food Chemistry 128 (2011) 214–217
Table 1
Table 2
Enzymatic synthesis of propyl gallate from methyl gallate and 1-propanol in aqueous
media. Reactions were performed as described in Section 2. Different pHs and
different concentrations of 1-propanol were tested.
Hydrolysis of different gallate esters catalysed by tannase immobilised on glyoxyl-
agarose. Reactions were carried out as described in Section 2 at pH 5.0 and 25 °C. (1)
Propyl gallate was also hydrolysed in the presence of 30% 1-propanol. (2) Propyl
gallate was also hydrolysed in the presence of 30% 1-propanol and in the presence of
10 mM gallic acid.
1-Propanol pH Activitya Initial ratio synthesis/
Final synthetic
yield (%)
(%)
hydrolysis
Substrate
Conditions
Activitya
30
50
30
50
5
7
0.19
0.16
0.48
0.42
1.2
1.5
1.0
1.2
45
55
40
45
Methyl gallate
Propyl gallate
Propyl gallate (1)
Propyl gallate (2)
Aqueous media
Aqueous media
30% 1-Propanol
30% 1-Propanol and gallic acid
0.42
0.50
0.14
0.036
a
Activity is expressed as the
lmol of reaction products (gallic acid plus propyl
a
gallate) produced per minute and per gram of immobilised biocatalyst.
Activity is expressed as the lmol of reaction product (gallic acid) produced per
minute and per gram of immobilised biocatalyst.
Fig. 1. Time-course of transesterification of tannic acid. The reaction was carried
out using 2 mM of tannic acid at pH 5.0 and 25 °C in the presence of 30% 1-
propanol. The final total concentration of propyl gallate (and gallic acid) was 20 mM
(100% of conversion). Squares: propyl gallate and circles: gallic acid.
Fig. 3. Time-courses of inactivation of tannase derivatives in the presence of 1-
propanol. Derivatives were incubated at pH 5 and 25 °C and 30% 1-propanol. The
remaining activity was measured in fully aqueous media at pH 7.0. Squares:
tannase derivatives obtained by multipoint covalent attachment on glyoxyl-
activated agarose; circles: tannase derivatives obtained by mild covalent immobi-
lisation on CNBr-activated agarose.
LPT was also able to catalyse the transesterification of tannic
acid by 1-propanol under the same experimental conditions. Syn-
thetic yields were very similar to those obtained with methyl gal-
late (45% of propyl gallate and 55% of gallic acid) (Fig. 1). The
experimental conditions (pH 5.0, 30% 1-propanol, 25 °C) were inert
from a chemical or microbiological point of view. Thus, both reac-
tion products, gallic acid and propyl gallate, were chromatograph-
ically pure (see Fig. 2).
After product separation, propyl gallate would be directly used
as a functional ingredient (natural antioxidant) in food technology,
and the by-product (pure gallic acid) could be utilised for multiple
applications.
3.2. Kinetic stability of propyl gallate
At the beginning of the synthesis, propyl gallate seems to be
partially hydrolysed to gallic acid and 1-propanol. In fact, synthetic
yields are slightly lower than those calculated from the initial syn-
thesis/hydrolysis ratio. However, when a reaction is reaching the
highest synthetic yield, propyl gallate becomes very stable. Its
hydrolysis is inhibited by gallic acid and 1-propanol (Table 2). In
addition, the presence of high concentrations of 1-propanol could
partially regenerate the propyl gallate.
Fig. 2. HPLC chromatogram of the final transesterification mixture. The chromatogram shows that two unique peaks of pure propyl gallate (RT = 12.8 min) and gallic acid
(RT = 1.8 min) were obtained.

G. Fernandez-Lorente et al. / Food Chemistry 128 (2011) 214–217
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4. Conclusions
Tannic acid is an inexpensive vegetal residue, and it was di-
rectly converted into propyl gallate (an interesting food antioxi-
dant) in aqueous media catalysed by immobilised tannase from
L. plantarum. Bearing in mind the natural raw material, reaction
media and biocatalyst, propyl gallate can be considered as a natu-
ral additive.
The best synthetic results were obtained at pH 5.0 in the pres-
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matographically pure propyl gallate and 55% of pure gallic acid.
Other tannases do not exhibit this interesting catalytic property.
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Acknowledgements
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This work has been supported by Grants AGL2008-01052, AGL-
2009-07625, Consolider INGENIO 2010 CSD2007-00063 FUN-C-
FOOD (CICYT), RM2008-00002 (INIA), and S-0505/AGR/000153
(CAM). J.A. Curiel is a recipient of pre-doctoral fellowships from
the I3P-CSIC Program and FPI-MEC, and Gloria Fernández-Lorente
is a recipient of Ramon y Cajal postdoctoral Contract.
Yu, X.-W., & Li, Y.-Q. (2006). Kinetics and thermodynamics of synthesis of propyl
gallate by mycelium-bound tannase from Aspergillus niger in organic solvent.
Journal of Molecular Catalysis B: Enzymatic, 40(1–2), 44–50.
Yu, X., Li, Y., & Wu, D. (2004). Enzymatic synthesis of gallic acid esters using
microencapsulated tannase: Effect of organic solvents and enzyme specificity.
Journal of Molecular Catalysis B: Enzymatic, 30(2), 69–73.
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