Synthesized tyrosyl hydroxyphenylacetate, a novel antioxidant, anti-stress and antibacterial compound

Article abstract of DOI:10.1016/j.procbio.2012.09.016

New tyrosyl ester derivative, a naturally occurring phenol with interesting biological properties, has been synthesized in good yield by a direct esterification of tyrosol (Ty) with p-hydroxyphenylacetic acid (p-HPA) using Candida antarctica lipase as a catalyst. The response surface methodology was used to modulate the effects of the enzyme amount (10-50 mg), the tert-butanol/hexane (v/v) ratio (0.16-0.84), the temperature (35-55 °C) and the reaction time (15-45 h) on the tyrosyl hydroxyphenylacetate (Ty-HPA) conversion yield. Under the optimal predicted conditions (enzyme amount: 10 mg, solvents volume ratio 0.16, reaction temperature; 45 °C and 34 h of incubation), a high conversion yield of 79.33 ± 4% was reached. The obtained ester was purified and characterized by NMR, LC/MS and FT-IR methods. ABTS free radical quenching potency demonstrated that the esterified tyrosol (Ty-HPA) was more effective than the natural separated antioxidants: Ty and p-HPA. Furthermore, when used at a non-cytotoxic concentration (100 μM), tyrosyl ester showed significant effectiveness in preventing iron-induced oxidative stress in blood cells compared to the two separated compounds. The antibacterial activity of Ty, p-HPA, mixed solution of Ty + p-HPA and Ty-HPA was performed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a micro-well dilution method. Compared to the separated substrates, synthesized ester exhibits the most antibacterial effect mainly against Gram+ bacteria.

Full text of DOI:10.1016/j.procbio.2012.09.016

Process Biochemistry 47 (2012) 2356–2364
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Synthesized tyrosyl hydroxyphenylacetate, a novel antioxidant, anti-stress
and antibacterial compound
Imen Aissaa, Mohamed Bouazizb, Fakher Frikhaa, Riadh Ben Mansourc, Youssef Gargouria,∗
a Laboratory of Biochemistry and Enzymatic Engineering of Lipases, ENIS route of Soukra, P.O. Box 1173, 3038, University of Sfax, Tunisia
b Electro-chemical Environmental Laboratory, ENIS route of Soukra, P.O. Box 1173, 3038, University of Sfax, Tunisia
c Unit of Biotechnology and Pathology, Higher Institute of Biotechnology of Sfax, P.O. Box “261”, 3038, University of Sfax, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2012
Received in revised form
20 September 2012
Accepted 23 September 2012
Available online 29 September 2012
New tyrosyl ester derivative, a naturally occurring phenol with interesting biological properties, has
been synthesized in good yield by a direct esterification of tyrosol (Ty) with p-hydroxyphenylacetic acid
(p-HPA) using Candida antarctica lipase as a catalyst. The response surface methodology was used to
modulate the effects of the enzyme amount (10–50 mg), the tert-butanol/hexane (v/v) ratio (0.16–0.84),
the temperature (35–55 ◦C) and the reaction time (15–45 h) on the tyrosyl hydroxyphenylacetate (Ty-
HPA) conversion yield. Under the optimal predicted conditions (enzyme amount: 10 mg, solvents volume
ratio 0.16, reaction temperature; 45 ◦C and 34 h of incubation), a high conversion yield of 79.33 4% was
reached. The obtained ester was purified and characterized by NMR, LC/MS and FT-IR methods. ABTS free
radical quenching potency demonstrated that the esterified tyrosol (Ty-HPA) was more effective than the
natural separated antioxidants: Ty and p-HPA. Furthermore, when used at a non-cytotoxic concentration
(100 ␮M), tyrosyl ester showed significant effectiveness in preventing iron-induced oxidative stress in
blood cells compared to the two separated compounds. The antibacterial activity of Ty, p-HPA, mixed
solution of Ty + p-HPA and Ty-HPA was performed by determining the minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC) using a micro-well dilution method. Compared to
the separated substrates, synthesized ester exhibits the most antibacterial effect mainly against Gram+
bacteria.
Keywords:
Tyrosol
Phenolic acid
Antioxidant
Response surface methodology
ABTS assay
Oxidative stress
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
of honeybee hives [7], has a high antioxidant activity. This product
presents also an anti-inflammatory [8], immunomodulatory, anti-
Natural phenolic acid compounds are numerous and widely dis-
tributed in the plant kingdom [1]. These molecules can be present
in considerable amounts in the human diet [2]. The phenolic acid
derivatives were revealed with a broad spectrum of pharmaco-
logical properties including anti-oxidation [3], anti-thrombosis [4],
anti-inflammatory [5], anti-viral and inhibition of human immuno-
deficiency virus (HIV) [6]. For example, it is well known that caffeic
acid phenethyl ester (CAPE), naturally extracted from the propolis
oral and anti-cancer activities [9]. Turkoglu et al. [10] have reported
that CAPE has a protective effect against neuronal damage in the
hippocampus of rats. Furthermore, the aromatic esters of hydroxy-
cinnamic acid derivatives such as phenylethyl- or tyrosyl-ferulate,
present in natural sources, have been found to exhibit an anti-
oxidant, anti-cancer, anti-HIVand anti-fungal/microbial activities
[11]. In addition, tyrosyl gallate, a chemical derivative of tyrosol,
could significantly suppress the cellular melanin formation without
cytotoxicity of melana cells [12].
Synthesis of the phenolic acid esters can be performed using
chemical or biological catalysts. The chemical synthesis is usually
carried out with basic or acidic catalysts under reflux. However,
these methods do not meet the requirements for food applica-
tions. CAPE was synthesized by direct esterification of caffeic acid
with phenethyl alcohol using dicyclohexyl carbodiimide as catalyst
[13]. A conversion yield of 38% was reached at the end of the reac-
tion. Some disadvantages of the chemical synthesizing processes
include the environmental pollution and the harm to the human
body caused by the use of chemical reagents. Nevertheless, the
use of enzyme synthesis offers the advantages of specificity, milder
Abbreviations:
Ty, tyrosol; p-HPA, p-hydroxyphenylacetic acid; Ty-HPA,
tyrosyl hydroxyphenylacetate; RSM, response surface methodology; LC/MS, liq-
uid chromatography coupled to mass spectrometry; FT-IR, Fourier transformed
infrared spectroscopy; NMR, nuclear magnetic resonance; ABT, S2,2-azino-bis-3-
ethylbenzothiazoline-6-sulfonic acid; TEAC, trolox equivalent antioxidant capacity;
RBC, red blood cells; MDA, Malondialdehyde; ROS, reactive oxygen species; TBA,
thiobarbituric acid; TCA, trichloroacetic acid; TEP, 1.1.3.3-tetraethoxypropane; MIC,
minimum inhibitory concentration; MBC, minimum bactericidal concentration;
MTT, 4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetrazolium bromide.
∗
Corresponding author. Tel.: +216 74675055; fax: +216 74675055.
E-mail address: ytgargouri@yahoo.fr (Y. Gargouri).
1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2012.09.016

I. Aissa et al. / Process Biochemistry 47 (2012) 2356–2364
2357
Table 1
Table 2
Levels of the factors tested in the Box-Behnken design.
The Box-Behnken design of RSM for optimization of the conversion yield of Ty-HPA.
Factors
Symbol
Coded levels
Experimental conditions Conversion yield (%)
−1
0
+1
50
0.84
55
45
Run
X1
X2
X3
X4
Observed
Predicted
Enzyme amount (mg)
Tert-butanol/hexane volumic ratio
Temperature (◦C)
X1
X2
X3
X4
10
0.16
35
15
30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
10
50
10
50
30
30
30
30
10
50
10
50
30
30
30
30
10
50
10
50
30
30
30
30
30
30
30
0.16
0.16
0.84
0.84
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.16
0.84
0.16
0.84
0.50
0.50
0.50
0.50
0.16
0.84
0.16
0.84
0.50
0.50
0.50
45
45
45
45
35
55
35
55
45
45
45
45
35
35
55
55
35
35
55
55
45
45
45
45
45
45
45
30
30
30
30
15
15
45
45
15
15
45
45
30
30
30
30
30
30
30
30
15
15
45
45
30
30
30
79.40
47.51
17.50
31.00
9.25
78.90
49.72
17.32
33.52
14.93
22.69
21.06
33.20
26.20
29.51
44.32
28.03
52.63
9.06
57.91
23.69
30.27
12.60
29.04
33.73
60.96
13.57
60.78
30.39
42.30
42.30
42.30
0.5
45
30
Reaction time (h)
24.00
17.72
36.86
25.40
28.50
46.00
29.50
51.20
12.45
55.20
25.80
32.34
17.59
25.40
33.00
66.30
14.40
61.30
26.40
41.00
42.50
43.40
reaction conditions and minimization of side effects. Recently, Chen
et al. [14] have reported the use of Novozyme 435 for the synthe-
sis of CAPE with a yield of 91.65% after 59 h at 69 ◦C. Same enzyme
was used recently by Croitoru et al. [15] for the synthesis of aro-
matic diesters of different sugar alcohols with 3-(4-hydroxyphenyl)
propionic acid.
In this study, a new phenethyl ester was synthesized by the
esterification of tyrosol (Ty) with the p-hydroxyphenylacetic acid
(p-HPA) using Novozyme 435 as a biocatalyst. A response sur-
face study using a Box Behnken design was set up to model
the relationship between the conversion yield of tyrosyl hydrox-
yphenylacetate (Ty-HPA) and the reaction factors (enzyme amount,
tert-butanol/hexane (v/v) ratio, temperature and reaction time).
In addition, chemico-biological activities of the newly synthesized
esters were investigated.
2. Material and methods
2.1. Chemicals
2.4. Chromatography conditions
The deuterated acetone, 4-hydroxyphenylacetic acid, 2,2-azino-bis-
3ethylbenzothiazoline-6-sulfonic acid (ABTS) and 4,5-dimethyl-thiazol-2-yl-
2,5-diphenyltetrazolium bromide (MTT) were purchased from Fluka (Suisse). The
n-hexane and 2-methyl-2-propanol were purchased from Prolabo (Paris, France).
Tyrosol, Candida antarctica lipase (Novozyme 435).
The identification of tyrosol, p-HPA and their ester was carried out using
HPLC system (Ultimate 3000, Dionex, Germany). The HPLC system was equipped
with a pump (LPG-3400SD), column oven and diode-array UV–vis detector (DAD-
3000RS). The output signal of the detector was recorded using Dionex ChromeleonTM
chromatography Data System. The separation was executed on an Inertsil ODS-
4 C18 colomn (5 ␮m, 4.6 mm × 150 mm) maintained at 35 ◦C. The flow rate was
1.5 mL/min, the injection volume was 20 ␮L and the detection UV wavelength was
set at 280 nm. The used mobile phase was 1‰ acetic acid in water (A) versus 0.5‰
acetic acid in acetonitrile (B) for a total running time of 8 min and the following
proportions of solvent B were used for the elution: 0–10% at 0–3 min; 10–30% at
3–5 min; 30–90% at 5–7 min and 90–10% at 7–8 min.
2.2. Experimental designs
2.2.1. Box-Behnken designs and response surface analysis
Box-Behnken designs of RSM were employed to optimize the four-selected fac-
tors (enzyme amount, tert-butanol/hexane volume ratio, temperature and reaction
time) for enhancing the conversion yield of Ty-HPA. The four-independent factors
were investigated at three different levels (−1, 0, +1) (Table 1) and the experimental
design used for the study is shown in Table 2.
2.5. Purification of tyrosyl ester
The conversion yield of Ty-HPA was fitted using a second-order polynomial
equation and a multiple regression of the data was carried out to obtain an empirical
model related to the factors [16]. The general form of the second-order polynomial
equation is:
The reaction mixture resulting from the esterification of p-HPA with tyrosol
contains a mixture of tyrosyl ester and residual substrates. After the removal of
the enzyme by centrifugation at 8000 rpm for 15 min, the mixture reaction was
dried under nitrogen and 100 mg was taken up in 1 mL chloroforme. The purifica-
tion of esters was achieved by chromatography on a silica gel 60 column (Merck)
(25 cm × 2 cm), previously equilibrated in chloroforme. Elution was carried using
chloroforme/methanol mixtures (90:10). The collected solvent fractions were ana-
lyzed by TLC using as the same mobile phase. The spots are revealed through
evaporated iodine. Purified fractions were pooled and solvents were evaporated
at 62 ◦C under vacuum.
ꢀ
ꢀ
ꢀ
Y = ˇ0
+
ˇiXi +
ˇiiXi2
+
ˇijXiXj
where Y is the predicted response, Xi and Xj are independent factors, ˇ0 is the
intercept, ˇi is the linear coefficient, ˇii is the quadratic coefficient and ˇij is the
interaction coefficient.
2.6. The LC/MS analysis
2.2.2. Data analysis and software
Design-expert, version 7.0 (STAT-EASE Inc., Minneapolis, USA) was used for the
experimental designs and statistical analysis of the experimental data. The analysis
of variance (ANOVA) was used to estimate the statistical parameters.
The LC/MS experiments were carried out with an Agilent 1100 LC system con-
sisting of degasser, binary pump, auto sampler, and column heater. The column
outlet was coupled to an Agilent MSD Ion Trap XCT mass spectrometer equipped
with an ESI ion source. Data acquisition and mass spectrometric evaluation were
carried out on a personal computer with Data Analysis software (Chemstations).
2.3. Esterification reactions
Production of tyrosyl derivative was performed by direct esterification of tyrosol
with p-HPA in screw-capped flasks. Equimolar of p-HPA to tyrosol ratio was dis-
solved in 6 mL of various tert-butanol/n-hexane volume ratios. The mixture was
stirred at different temperatures in an orbital shaker at 200 rpm and in presence of
Candida antarctica lipase (Novozyme 435). Controls were run in parallel, under the
same conditions, without enzyme addition. Aliquots of the mixture reaction were
withdrawn at different time of incubation and filtered to be used for HPLC analysis.
The conversion yield of tyrosyl derivative was calculated as the ratio of number of
mol of synthesized compound (determined by a standard range previously estab-
lished) per total number of mol of tyrosol.
2.7. NMR and FT-IR experiments
1H NMR and 13C NMR spectra were recorded in deuterated acetone on a Bruker
AV300 spectrometer operating at 300 MHz and 75 MHz respectively. IR spectra were
recorded on FT/IR-410 (JASCO).
Ty-HPA. Colorless oil. 1H NMR (300 MHz, Acetone-d6): 8.16 (1H (H6 ), s); 6.90
ꢀ
(2H (H5), d, J = 9 Hz); 6.93 (2H (H5 ), d, 3J = 9 Hz); 6.62 (2H (H4), d, 3J = 8.5 Hz);
ꢀ
6.64 (2H (H4 ), d, 3J = 8.5 Hz); 4.05 (2H (H1), t, CH2
O
C
C
O); 3.36 (2H (H2 ),
ꢀ
ꢀ
s, CH2 OCOCH2); 2.67 (2H (H2), t, CH2 CH2
Acetone-d6): 34.84 (C2), 40.79 (C2 ), 66.05 (C1), 116.01 (C5 + C5 ), 126.16 (C3), 129.6
O
O). 13C NMR (75 MHz,
ꢀ
ꢀ

2358
I. Aissa et al. / Process Biochemistry 47 (2012) 2356–2364
OH
6'
OH
OH
OH
6
6
6'
5'
4'
5'
4'
5
4
5
Novozyme 435
Lipase
+
4
3
3'
2'
3'
2'
3
O
2
HO
1'
1'
OH
2
1
1
O
O
Tyrosyl hydroxyphenyl acetate
(Ty-HPA)
Tyrosol
(Ty)
p-hydroxyphenylacetic acid
(p-HPA)
Scheme 1. Esterification reaction between Tyrosol and p-hydroxyphenyl acetic acid.
ꢀ
ꢀ
ꢀ
ꢀ
(C3 ), 130.75 (C4 + C4 ), 156.88 (C6 + C6 ); 172.13 (C1 , C O). MS: mass calculated
272.31 for C16H16O4 found m/z = 290 [M+NH4+], 272 [M+], 191 [M−C5H5O] and
121[M−p-HPA-H2O]. IR (liquid) cm−1: 3256–3023(HO-Ø), 2952 (C H), 1703(C O),
1598 (C C) and 1220 (C O).
3. Results and discussion
A response surface design was performed to optimize the enzy-
matic synthesis of tyrosyl ester by a direct esterification of tyrosol
with p-hyroxyphenylacetic acid using Novozyme 435 as a cata-
lyst (Scheme 1). Several conditions were tested including various
enzyme amounts, solvents’ ratios, temperatures and reaction times.
In a preliminary study, we have shown that the addition of hexane
to the reaction medium was necessary to improve the conversion
yield and the stability of the immobilized lipase. Also, we have
found that neither the increase of reaction time up to 45 h, nor the
2.8. Measurement of the trolox equivalent antioxidant capacity (TEAC)
The ABTS radical-scavenging activity was determined according to Re et al. [17].
The ABTS radical cation was prepared by reacting an aqueous solution of ABTS
(7 mM) with potassium persulfate (2.45 mM, final concentration) which was kept
in the dark at 25 ◦C for 12–16 h. The obtained solution was diluted in ethanol to
an absorbance of 0.70 ( 0.020) at 734 nm before use. 10 ␮L of trolox or sample
in ethanol were mixed with 990 ␮L of this diluted solution and the absorbance
was determined at 734 nm and 30 ◦C, 6 min after initial mixing. Appropriate sol-
vent blanks were run in each assay. The extent of decolorization was estimated by
monitoring the reduction of the absorbance at 734 nm. The anti-oxidant activity
was determined as a function of compounds and calculated relative to the equiva-
lent trolox concentration. The activity of each antioxidant was determined at three
concentrations, within the range of the dose–response curve of trolox. The radical-
scavenging activity was expressed as the trolox equivalent antioxidant capacity
(TEAC) defined as mM of trolox.
˚
addition of water or a crude molecular sieve 4 A (up to 5%, w/w) at
the beginning of reaction improved the conversion yield. In the light
of these results, enzyme amount, tert-butanol/hexane (v/v) ratio,
temperature and reaction time were chosen as the most effective
operating variables on the response.
3.1. Analysis of variance and validation of the models
2.9. Prevention of oxidative stress in human blood
Based on this preliminary study, RSM using Box Behnken
was applied to determine the optimal levels of the four-selected
variables (enzyme amounts, tert-butanol/hexane volume ratio,
temperature and reaction time) which significantly influenced the
conversion yield. The respective low and high levels with the coded
levels for the four variables are defined in Table 1. A total of 27
runs with different combination of enzyme amounts (X1), solvent’s
ratio (X2), temperature (X3) and reaction time (X4) was designed in
Table 2.
Blood was collected from healthy donors in centrifugal tubes containing 1 mL
of citrate as anti-coagulant. After centrifugation 10 min at 1500 rpm, the sera were
discarded and the red cells were collected and diluted to 1/4. 100 ␮L was added to
400 ␮L of water and subjected to different treatments (with or without the tested
compounds). Oxidative stress was induced by adding 100 ␮mol of Fe2+ to cells sus-
pension and incubated at 37 ◦C. After 1 h, an equal volume of TBA reagent (0.8%
of TBA and 15% TCA in HCl 0.25 N) was added. The mixture was heated at 95 ◦
C
during 15 min. After centrifugation at 3000 rpm during 10 min, the optical density
was recorded at 532 nm. Values were reported to a calibration curve of 1.1.3.3-
tetraethoxypropane (1.1.3.3 TEP).
The observed and predicted responses of the twenty-seven
experiments are also presented in Table 2. The experimental results
were analysed by standard ANOVA and the Box Behnken design was
fitted with the second order polynomial equation:
2.10. Determination of the minimum inhibitory concentration (MIC) and the
minimum bactericidal concentration (MBC)
The antibacterial activities of Ty, p-HPA, Ty + p-HPA (taken together at the
same concentration) and Ty-HPA were tested against Bacillus (B.) cereus, B. subtilis,
Enterococcus (E.) faecalis, Staphyloccocus (S.) aureus, S. xylosus, Enterobacter (E.) cloa-
cae, Echerichia (E.) coli, Klebsielle (K.) pneumonia and Pseudomonas (P.) aerigenosa
using Luria-Bertani (LB) medium. The minimum inhibitory concentration (MIC)
values, which correspond to the lowest compound concentration that completely
inhibits the growth of microorganisms, were determined by a micro-well dilution
method as previously described by Eloff [18] using 3-(4,5-dimethyl-thiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT). The inoculum of each bacterium was
prepared and the suspensions were adjusted to 106 CFU/mL. All the compounds were
dissolved in 100% ethanol, and then dilutions series were prepared in a 96-well plate,
ranging from 6.25 ␮g/mL to 4 mg/mL. Each well of the microplate contains 175 ␮L
of the growth medium, 5 ␮L of inoculum and 20 ␮L of the diluted sample extract.
Ethanol is used as a negative control. The plates were incubated at 37 ◦C for 24 h,
then 40 ␮L of MTT, at a final concentration 0.5 mg/mL freshly prepared in sterile
water, was added to each well and incubated for 30 min. The change to purple color
indicated that the bacteria were biologically active. The MIC was taken where no
change of colour of MTT was observed in the well.
yˆ = 42.3 − 3.25X1 − 19.45X2 + 4.98X3 + 4.16X4 − 3.43X12
+ 5.99X22 − 12.47X32 − 6.86X42 + 11.35X1X2 + 5.59X1X3
−4.9X1X4 + 2.34X2X3 + 4.25X2X4 + 1.1X3X4
where X1, X2, X3 and X4 are the coded factors of enzyme amount,
solvent’s ratio, temperature and reaction time respectively.
As shown in Table 3, the fit of the model was checked by the
coefficient of determination R2 which was equal to 0.9753 indicated
that 97.53% of the variability in the response could be explained
by the model. The statistical significance of the model equation
was evaluated by the F-test for ANOVA. The model F-value of 33.84
implied the model was significant. There was only a 0.01% chance
that the model F-value could occur due to noise. The P-value was
also very low (P < 0.0001) indicating the significance of the model.
The lack of fit F-value of 12.95 implied that there was a 7.38% chance
that the lack of fit F-value could occur due to noise.
For the determination of minimum bactericidal concentration (MBC), a portion
of liquid from each well that showed no change in color will be placed on solid LB
and incubated at 37 ◦C for 24 h. The lowest concentration that yielded no growth
after this sub-culturing will be taken as the MBC [19]. All experiments were done in
duplicate.

I. Aissa et al. / Process Biochemistry 47 (2012) 2356–2364
2359
Table 3
Statistical analysis of the model (ANOVA).
Source
Sum of squares
Degrees of freedom
Mean square
F value
p-value Prob > F
Model
7630.48
193.27
190.33
2.94
14
12
10
2
545.03
16.11
19.03
1.47
33.84
<0.0001a
Residual
Lack of fit
Pure error
Cor total
12.95
0.0738
7823.75
26
R-squared
0.975
√
Estimated standard deviation is equal to Std. Dev. = 193.27/12 = 4.01.
a
Statistically significant at 95% of confidence level.
3.2. Graphical interpretation of the response surface model
essential water from the enzyme surface, leading to an insuf-
ficiently hydrated enzyme molecule and in consequently to a
decrease of the enzymatic activity [21]. The use of solvent with
a log P > 3 (hexane) shows better reaction rates. Therefore, the
hydrophobic solvents preserve the catalytic activity without dis-
turbing the micro-aqueous layer of the enzyme [22]. Furthermore,
the variation in the bioconversion yield and enzyme activity with
the organic solvents’ ratio may be due to the substrate–solvent
interactions which could affect the partition coefficients of sub-
strates as well as products and subsequently the overall activity of
the enzyme-catalyzed reaction [23].
Fig. 2 represents the effect of varying solvents’ ratio and reaction
time at 45 ◦C in presence of 30 mg enzyme amount. At low sol-
vents volume ratio (0.16), the conversion yield passes from 66.3%
to 61.3% at (15 h) and (45 h) reaction time respectively. Under these
conditions, the maximum conversion yield was reached at 35 h
The response surface curves are plotted to explain the interac-
tion of the variables and to determine the optimum level of each
variable to reach a maximum conversion yield. The response sur-
faces curves are shown in Figs. 1–3. Each figure demonstrates the
effect of two factors while the others were fixed at zero level.
We can notice that the increasing of the solvent volume ratio
at any given enzyme amount (Fig. 1) or at any given time (Fig. 2)
leads to a decrease in the conversion yield. These results could
be explained by the negative effect of the hydrophilic solvent
on the enzyme activity. The changes in the enzyme microen-
vironment induced by the nature of the organic solvents could
explain the low bioconversion yield in the polar medium [20]. In
fact, hydrophilic solvents with log P < 2.5 might strip off even the
Fig. 1. Contour plots and response surface curve for the conversion yield of Ty-HPA
showing the interaction between the enzyme amount and solvent’s volume ratio.
Temperature and time are equal to 45 ◦C and 30 h respectively.
Fig. 2. Contour plots and response surface curve for the conversion yield of Ty-HPA
showing the interaction between the solvent’s volume ratio and the reaction time.
Temperature and enzyme amount are equal to 45 ◦C and 30 mg respectively.

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I. Aissa et al. / Process Biochemistry 47 (2012) 2356–2364
Fig. 4. Production of Ty-HPA during esterification reaction. Reaction conditions:
enzyme amount of 10 mg, tert-butanol/hexane volume ratio of 0.16 and temperature
of 45 ◦C. The tyrosol ester yield was estimated using HPLC system.
hence its availability for the reaction [27]. The presence of high
protein-support interactions as a result of an excess of the enzyme
could alter its active conformation and hence it’s catalytic efficiency
[28]. Steric hindrance of the enzyme active site may contribute to a
decrease in the enzymatic activity at high enzyme concentrations
[29].
3.3. Optimal reaction conditions
The optimal values of the four variables predicted by the
model are X1 = 10.05 mg, X2 = 0.16, X3 = 44.76 ◦C and X4 = 34.49 h
corresponding respectively to enzyme amount, solvent’s ratio, tem-
perature and reaction time. The maximum predicted conversion
yield was 79.7 3.18%. Under these optimal conditions, the time
course of the esterification reaction between the Ty and p-HPA was
presented in Fig. 4. A good correlation was found between predicted
and experimental values implying that empirical models derived
from response surface methodology can adequately describe the
relationship between the factors and their influence on the tyrosyl
ester synthesis. The level of Ty-HPA increased rapidly to reach its
maximal value at 34 h. The experimental conversion yield of Ty-
HPA (79.33 4%) was very close to the predicted estimated value
(79.7 3.18%) at a reaction time of 34 h.
Fig. 3. Contour plots and response surface curve for the conversion yield of Ty-HPA
showing the interaction between the enzyme amount and temperature. Solvent’s
volume ratio and reaction time are equal to 0.5 and 30 h respectively.
of incubation time. The same enzyme was used in the synthe-
sis of aromatic esters of hydroxycinnamic acid derivatives, such
as phenylethyl- or tyrosyl-ferulate by Stevenson et al. [11]. The
authors found that the reaction rates reached the equilibrium after
a long incubation time (200 h).
3.4. Structure determination of Ty-HPA ester
Tyrosol contains two hydroxyl groups in its structure, and there-
fore two esterified derivatives with p-HPA were expected. HPLC
analysis of the reaction mixture after 34 h (Fig. 5) showed that
tyrosol and p-HPA were transformed by enzymatic reaction to a
new product eluted at a retention time of 6.51 min.
Fig. 3 shows the effect of varying enzyme amount and the
reaction temperature on the esterification yield checked at a con-
stant solvents’ ratio of 0.5. It can be seen from Fig. 3 that at any
given enzyme amount, the conversion yield increases when the
temperature passes from 35 to 45 ◦C and decreases above 45 ◦C.
Considering the fact that Novozym 435 possesses high thermosta-
bility in organic solvents [24], this result can be related to the
decrease in equilibrium constants at higher reaction temperatures
[25]. The same behavior was previously described by Rahman et al.
[25] for the synthesis of dioleyl succinate ester by Novozyme 435.
These authors showed that the maximum conversion yield was
reached at 40.2 ◦C then the esterification yield decreased with an
increase in the temperature from 40.2 to 65.0 ◦C.
We can also conclude, from Fig. 3, that the conducted reaction
with a low amount of enzyme (10 mg) at 45 ◦C leads to an enhance-
ment of the conversion yield. However, the increase of the enzyme
concentration (50 mg) is accompanied by a significant decrease of
the conversion yield. This phenomenon has also been reported by
Zheng et al. [26] for the lipase-catalyzed transesterification of ethyl
ferulate with triolein. These results may be explained by a mass
transfer limitation at a high enzyme concentration, which could
affect the diffusion of the substrate to the enzyme active site and
On the basis of the NMR (Fig. 6), LC/MS (Fig. 7) and FT-IR data,
it was possible to determine the structure of the new compound.
With regard to 13C NMR data, the spectrum of the esterified tyrosol
showed signals at ı 172 ppm, which was attributed to a carbon
ester function (C1 ) (Fig. 6A) was confirmed by 1H NMR. Indeed,
ꢀ
with comparison to the 1H NMR spectrum of tyrosol [30], no sig-
nificant change in chemical shift was observed, only a downfield
at 4.05 ppm for the triplet (2H) observed at 3.8 ppm in 1H NMR
of tyrosol suggesting that ester function was linked to the methy-
lene group C1 (Fig. 6B). For the aromatic protons of the tyrosol and
its esterified form, no modification of chemical shift was observed.
It follows that by using Novozyme 435 as biocatalyst, tyrosol was
esterified on the primary hydroxyl group.
The LC/MS analysis in positive mode exhibited a molecular ion at
m/z = 290 [M+NH4+] with intermediate fragments at m/z = 272 [M+],
191 [M−C5H5O] and 121[M−pHPA-H2O]. The MS/MS experiments
focusing on the fragment generated from the peak in m/z = 290
[M+NH4+] revealed a fragment at m/z = 272 corresponding to the

I. Aissa et al. / Process Biochemistry 47 (2012) 2356–2364
2361
2 700
2 700
WVL:280 nm
WVL:280 nm
3-6.510
A
B
2 000
1 500
1 000
500
2 000
1 500
1-4.977
2-5.193
1 000
500
0
1-4.973
2-5.180
0
0,00
2,00
2.00
4,00
4.00
6,00
6.00
8,00
8.00
0,00
2,00
2.00
4,00
4.00
6,00
6.00
8,00
8.00
0.00
0.00
Retention Time (min)
Retention Time (min)
Fig. 5. HPLC chromatograms of the reaction medium before and after synthesis of Ty-HPA. The separation was made on C18 reverse-phase HPLC. Flow 1.5 mL/min, and UV
detection was at 280 nm. (A) Time of esterification = 0 h. (B) Time of esterification = 34 h. 1: Tyrosol. 2: p-HPA. 3: Ty-HPA. Synthesis was performed under optimal conditions.
synthesized compound and the pseudo molecular ion at m/z = 121
attributed to the tyrosol ion.
3.6. Biological antioxidant activity in human cell cultures
The formation of Ty-HPA was confirmed by comparing the FT-
IR spectra of tyrosol and its ester. The FT-IR spectra showed a peak
at 1703 cm−1 attributed to a vibration of carbonyl group and peak
at 2952 cm−1 attributed to C H vibration of methylene groups. In
addition, a large band around 3256–3023 cm−1 was attributed to
the hydroxyl group linked to the aromatic ring. Other peaks were
detected at 1598 and 1220 cm−1 attributed to C C and C O respec-
tively. The chemical change in functional groups of the simples
indicated the formation of Ty-HPA ester. To our knowledge, no
reports on the chemical or enzymatic synthesis of such phenyl ethyl
ester have been reported.
The investigation of the biological antioxidant activity of Ty, p-
HPA and Ty-HPA was carried out on freshly isolated human red
blood cells (RBC). RBC were incubated with or without adding
the compounds at different concentrations. Oxidative stress was
induced by adding 100 ␮M Fe2+ solution (as Fe2SO4) in PBS for 1 h.
Malondialdehyde (MDA) production, a lipid peroxidation marker,
was evaluated and compared with control cells. As shown in
Fig. 8, a significant protection against ROS (reactive oxygen species)
inducing damage was obtained in the presence of tyrosyl hydrox-
yphenyacetate (Ty-HPA) at a concentration of 100 ␮g/mL. The
addition of Ty and p-HPA at the same concentration failed to
decrease the MDA level as compared to control cells. This suggests
that the tyrosyl ester is more efficient than the two separated sub-
strates used in synthesis. These results are in correlation with those
found by Stevenson et al. [11]. In fact, the authors reported that
the esterification of some phenolic acids can apparently increase
the strength of protection of Jurkat cells from hydrogen peroxide
mediated cytotoxicity. This could be a result of higher hydropho-
bicity increasing absorption of the esters into the cell membrane
and resulting in greater bioavailability.
3.5. The ABTS assay
A widely used method for measuring the radical-scavenging
activity of antioxidants is the ABTS assay, where the activity
toward a stable free radical, 2,2-azino-bis-3ethylbenzothiazoline-
6-sulfonic acid (ABTS•+), is evaluated. The radical-scavenging activ-
ities of the evaluated antioxidants Tyrosol, p-hydroxyphenylacetic
acid and their corresponding ester and BHT are summarized in
Table 4. Results are expressed as Trolox equivalent antioxidant
capacity (TEAC, mM). The TEAC measured with synthesized com-
pound is higher than the value obtained for each substrate tyrosol
or phenolic acid. The substrates taken together in the same solu-
tion at a final concentration of 2 mg/mL presents a TEAC value
lower than the synthesized ester (data not shown). Their scav-
enging activity of ABTS radicals decreased in the following order
BHT > Ty-HPA >Ty> p-HPA with all values significantly different at
p < 0.05. This sequence indicates that the ABTS radical scavenging
activity of the tested compounds is to their hydrogen-donating abil-
ity. It is generally assumed that the ability to act as an hydrogen
donor and the inhibition of oxidation are enhanced by increasing
the number of hydroxyl groups in the phenol ring [31].
3.7. Determination of the minimum inhibitory concentration
(MIC) and the minimum bactericidal concentration (MBC)
The antibacterial activity of Ty, p-HPA, Ty + p-HPA and the
synthesized ester Ty-HPA was checked against Gram-positive (B.
cereus, B. subtilis, E. faecalis, S. xylosus, S. aureus) and Gram-negative
(E. cloacae, K. pneumoniae, E. coli, P. aerigenosa) bacteria by the
determination of MIC and MBC values (Table 5). As it can be seen
from Table 5, Ty has not a significant inhibition activity against all
bacteria tested (MIC and MBC values are ≥ 4 mg/mL). Compared to
Ty, p-HPA and mixed solution of Ty + p-HPA, the synthesized ester
exhibits the most effect against the bacteria tested, in particular the
Gram+ ones. With Gram+ bacteria, the MIC values are ranged from
0.5 to 0.8 mg/mL and the MBC values are between 0.5 and 2 mg/mL.
Gram− bacteria appeared to be less sensitive to the tested
compounds. Previous studies [32] reported that Gram+ bacte-
ria presented higher sensitivity than Gram− bacteria to various
polyphenols. This higher resistance can be related to the com-
position of the cell-wall membrane [32]. As it can be seen from
Table 5, MIC values showed that Ty-HPA exhibits a similar or a
slight higher activity against Gram− bacteria than the separated
substrates. Nevertheless, except E. cloacae strain, the obtained
MBC values confirmed that the synthesized ester (Ty-HPA) has
more bactericidal effect against Gram− bacteria than the separated
Table 4
Reducing antioxidant power of tyrosol, p-HPA and their derivative. Evaluation by
ABTS assay.
Compounds
TEAC (mM)a
Tyrosol
p-HPA
Ty-HPA
BHT
1.22 0.03
0.87 0.015
1.56 0.006
2.15 0.001
a
Each value is the mean of triplicate measurements standard deviations
(p < 0.05). Results are expressed as Trolox equivalent antioxidant capacity (TEAC)
in units of mmol Trolox/L.

2362
I. Aissa et al. / Process Biochemistry 47 (2012) 2356–2364
Fig. 6. The 1H NMR (A) and 13C NMR (B) spectra of synthesized ester.
Table 5
Antimicrobial activities of Ty, p-HPA, Ty + p-HPA and Ty-HPA.
Strain
Gram
CMI (mg/mL)
CMB (mg/mL)
Ty
p-HPA
(Ty + p-HPA)a
Ty-HPA
Ty
p-HPA
(Ty + p-HPA)a
Ty-HPA
Bacillus cereus
Bacillus subtilis
+
+
+
+
>4
4
4
>4
>4
>4
>4
>4
>4
2
1
1
2
2
2
2
1
1
2
1
2
2
2
2
2
1
1
0.8
0.5
0.8
0.5
0.5
1
1
1
1
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
4
4
4
4
4
>4
>4
>4
4
4
2
2
4
4
>4
>4
2
1
0.8
0.5
0.8
4
2
2
2
Enterococcus faecalis
Staphylococcus aureus
Staphylococcus xylosus
Enterobacter cloacae
Klebsielle pneumoniae
Escherchia coli
+
−
−
−
−
Pseudomonas aerigenosa
a
Taken together at the same concentration.

I. Aissa et al. / Process Biochemistry 47 (2012) 2356–2364
2363
+MS, Line, 15.1min (#671), 100%=40192352
7
289.7
x10
[M+NH4] +
4
3
2
1
0
[M-pHPA-H2O] +
120.9
[M-C5H5O] +
191.9
[M+H] +
[M-pHPA] +
272.4
310.8
300
138.0
223.9
238.7
50
100
150
200
250
350
400
m/z
Intens.
+MS2(289.9), Line, 14.5-15.0min (#644-#664), 100%=16788343
1.[M-pHPA-H2O] +
7
x10
120.8
1.5
1.0
0.5
0.0
[M+H] +
272.6
100
200
300
400
500
600
700
m/z
Fig. 7. LCMS (+ve mode) profile of peak identified as Ty-HPA.
that the ester was more efficient than the tyrosol or the phe-
nolic acid. The synthesis of phenol derivatives via esterification
of the hydroxyl functions with aliphatic phenols can be used
as a tool to increase their antioxidant activity and to improve
therefore their stability and biological function. Moreover, this
aromatic analogue could be used in cosmetic and pharmaceutical
fields.
Conflict of interest
The authors declare that they have no conflict of interest.
Fig. 8. Ability of hydroxytyrosol and acylated derivatives to reduce iron-induced
oxidative stress within blood cells. C-untreated control cells, C-Ox iron-stressed or
positive control cells, Ty, p-HPA and Ty-HPA refer to cells treated with both FeSO4
and the Ty, p-HPA and Ty-HPA corresponding compound. Statistical significance at
p < 0.0001 was calculated relatively to the positive control.
Acknowledgements
We are grateful to Dr. Noureddine Raouafi (FST) for the RMN
analysis, Dr. Ahlem Kabadou (FSS) for FT-IR analysis, Miss Lobna
Jlail (CBS) for LC/MS analysis, Pr. Sami Sayadi for supporting us
with tyrosol and Dr. Nabil Miled, Mr. Mohamed Sellami and Miss
Nadia Kharrat for their fruitful discussion during the prepara-
tion of this manuscript. The authors are indebted to Professor Mr.
Riadh Koubaa from the “OPEN School Center” a language Center,
Sfax, Tunisia, who reviewed this manuscript. This work received
financial support from the Ministry of Higher Education, Scientific
Research in Tunisia.
substrates. These results indicate that we have enhancing signif-
icantly the antimicrobial activity of the Tyrosol and p-HPA after
esterification of these compounds. The potent antibacterial activ-
ity of the synthesized compound could be related to the presence
of two hydroxyl group at the p-positions of the benzene rings of Ty-
HPA ester. These results are in concordance with those reported by
Fu et al. [33]. In fact, they have found that caffeic acid anilides with
electron donating groups at p-position of benzene ring have better
antibacterial activities.
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