Enantioselective synthesis of (S)-naproxen using immobilized lipase on chitosan beads

Article abstract of DOI:10.1002/chir.22689

S-naproxen by enantioselective hydrolysis of racemic naproxen methyl ester was produced using immobilized lipase. The lipase enzyme was immobilized on chitosan beads, activated chitosan beads by glutaraldehyde, and Amberlite XAD7. In order to find an appropriate support for the hydrolysis reaction of racemic naproxen methyl ester, the conversion and enantioselectivity for all carriers were compared. In addition, effects of the volumetric ratio of two phases in different organic solvents, addition of cosolvent and surfactant, optimum pH and temperature, reusability, and inhibitory effect of methanol were investigated. The optimum volumetric ratio of two phases was defined as 3:2 of aqueous phase to organic phase. Various water miscible and water immiscible solvents were examined. Finally, isooctane was chosen as an organic solvent, while 2-ethoxyethanol was added as a cosolvent in the organic phase of the reaction mixture. The optimum reaction conditions were determined to be 35?°C, pH?7, and 24?h. Addition of Tween-80 in the organic phase increased the accessibility of immobilized enzyme to the reactant. The optimum organic phase compositions using a volumetric ratio of 2-ethoxyethanol, isooctane and Tween-80 were 3:7 and 0.1% (v/v/v), respectively. The best conversion and enantioselectivity of immobilized enzyme using chitosan beads activated by glutaraldehyde were 0.45 and 185, respectively.

Full text of DOI:10.1002/chir.22689

Received: 4 November 2016
DOI: 10.1002/chir.22689
Revised: 11 December 2016
Accepted: 16 December 2016
R E G U L A R A R T I C L E
Enantioselective synthesis of (S)‐naproxen using immobilized
lipase on chitosan beads
Saeedeh L. Gilani1 | Ghasem D. Najafpour1
| Hamid D. Heydarzadeh2 | Aliakbar Moghadamnia3
1 Faculty of Chemical Engineering, Babol
Abstract
Noshirvani University of Technology, Babol,
S‐naproxen by enantioselective hydrolysis of racemic naproxen methyl ester was
produced using immobilized lipase. The lipase enzyme was immobilized on chito-
san beads, activated chitosan beads by glutaraldehyde, and Amberlite XAD7. In
order to find an appropriate support for the hydrolysis reaction of racemic naproxen
methyl ester, the conversion and enantioselectivity for all carriers were compared. In
addition, effects of the volumetric ratio of two phases in different organic solvents,
addition of cosolvent and surfactant, optimum pH and temperature, reusability, and
inhibitory effect of methanol were investigated. The optimum volumetric ratio of
two phases was defined as 3:2 of aqueous phase to organic phase. Various water
miscible and water immiscible solvents were examined. Finally, isooctane was cho-
sen as an organic solvent, while 2‐ethoxyethanol was added as a cosolvent in the
organic phase of the reaction mixture. The optimum reaction conditions were deter-
mined to be 35 °C, pH 7, and 24 h. Addition of Tween‐80 in the organic phase
increased the accessibility of immobilized enzyme to the reactant. The optimum
organic phase compositions using a volumetric ratio of 2‐ethoxyethanol, isooctane
and Tween‐80 were 3:7 and 0.1% (v/v/v), respectively. The best conversion and
enantioselectivity of immobilized enzyme using chitosan beads activated by glutar-
aldehyde were 0.45 and 185, respectively.
Iran
2 Faculty of Petroleum and Petrochemical
Engineering, Hakim Sabzevari University,
Sabzevar, Iran
3 Department of Pharmacology and
Physiology, School of Medicine, Babol
University of Medical Sciences, Babol, Iran
Correspondence
Ghasem D. Najafpour, Faculty of Chemical
Engineering, Noshirvani University of
Technology Babol, Iran, Mazandaran, Babol.
Email: najafpour@nit.ac.ir;
najafpour8@yahoo.com
KEYWORDS
Amberlite XAD7, chitosan beads, enzymatic hydrolysis, lipase enzyme, naproxen, surfactant
1 | INTRODUCTION
widely used as NSAIDs for the treatment of arthritis and
body pains. The high activity is exhibited by the (S)‐config-
Enantiomerically pure compounds are prominently being used
inpharmaceuticalsandagrochemicalproduction. Thereasonis
thatoneofthetwoenantiomersisoftenmorebiologicallyactive
than the other or may cause undesired side effects.1,2 In addi-
tion, based on registration of new chiral drugs, the production
of enantiomerically pure compounds are permitted. This fact
has led to great efforts to develop efficient methods to obtain
enantiomerically pure compounds in recent years.3-5
uration at the chiral center (28‐fold); (S)‐naproxen has the
favorable therapeutic effect. In contrast, the (R)‐naproxen is
inactive and also can cause side effects such as liver dis-
ease.5-8 Subsequently, finding novel techniques for produc-
tion of enantiomerically pure (S)‐naproxen is important for
researchers.
Use of enzymes as enantioselective biocatalysts is a valu-
able method for obtaining optically pure compounds.9
Lipases play the most important role among the enzymes that
have been widely employed for the resolution of racemic
compounds.2,3,5,10 Understanding the structure and function
Nonsteroid antiinflammatory drugs (NSAIDs) are rated
as the top chiral drugs. (S)‐naproxen is an important member
of the family of 2‐aryl propionic acid derivatives, which are
Chirality. 2017;1–11.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

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GILANI ET AL.
of lipase is crucial to process it in its active form. Lipase
enzyme is soluble in water, while its substrates are soluble
in organic phase.
The catalytic site of lipase (the triad of serine, histidine,
and aspartic acid) is hidden within the structure of the mono-
meric protein, which is exposed at the organic–water inter-
face called interfacial activities.
This mechanism might may propose the presence of a cap
(lid) that means, when cover is closed causes the catalytic site
makes the enzyme inactive; in contrary, presence of the inter-
face as open cover should reveal the active site.11-14
However,specialcharacteristicoftheindustrialapplication
for free enzymes such as good stability, activity, and selectivity
under conditions far from the physiological environment is
often restricted. To overcome such restrictions, one of the most
successful methods is immobilization of the enzyme. Immobi-
lization of the enzyme can create a heterogeneous biocatalyst
that is easily recovered from the reaction mixture, reutilized,
and stabilized at the reaction severe conditions. The selection
of appropriate support is the first requirement when applying
an immobilized enzyme.13,15-18 Natural biopolymers such as
cellulose, starch, dextran, agarose, and chitosan were exten-
sively applied as carriers for the immobilization of enzymes.
From this group, chitosan is the most desired support for
enzyme immobilization due to its excellent properties, such as
biocompatibility, nontoxicity, biodegradability, physiological
inertness, and high affinity to proteins.13,19-23 A large number
ofresearchhasstudiedthedifferentsupportsforenzymaticpro-
duction of a chiral drug2,3,24-27; but no literature reports exist
indicating chitosan beads as an enzyme carrier for synthesis
of enantiomerically pure naproxen.
beads and activated chitosan beads. In addition, the effect of
thevolumetricratiooftwophasesanddifferentorganicsolvent,
additional cosolvent and surfactant, optimum pH and tempera-
ture, reusability on conversion, and enantioselectivity toward
optically pure S‐naproxen production were investigated.
2 | MATERIALS AND METHODS
2.1 | Materials
Lipase from Candida rugosa (Type VII), low‐viscosity chito-
san, and glutaraldehyde (50% solution), and Amberlite
XAD7 were acquired from Sigma (St. Louis, MO). Racemic
naproxen were purchased from Alborz Bulk Pharmaceutical
(Tehran, Iran). High performance liquid chromatography
(HPLC)‐grade organic solvents were supplied by Merck
(Darmstadt, Germany) as the HPLC eluent without further
purification or drying. All other chemicals used in this work
were analytical or reagent grade, from Merck.
2.2 | Analytical methods
Fourier transform infrared (FT‐IR) spectra were analyzed by
KBr pellets on a FT‐IR spectrometer (WQF ‐ 510A, China).
HPLC (Knauer, Germany) were carried out using a Vertex
Plus Column 250 × 4.6 mm Eurocel 01, 5 μm with pr‐column
(Knauer, Germany) at 25 °C. R/S ratios of naproxen methyl
ester were determined using n‐hexane/2‐propanol/
trifluoroacetic acid (100/2/0.2%, v/v/v) as the mobile phase
at the flow rate of 1 mL/min, and UV detection was carried
out at a wavelength of 254 nm. R/S ratios of naproxen were
determined using n‐hexane/2‐propanol/trifluoroacetic acid
(90/10/0.1%, v/v/v) as the mobile phase at the flow rate of
1 mL/min; UV detection was conducted at a wavelength of
230 nm. The enantioselectivity was expressed as the enantio-
meric ratio (E), which is calculated from the conversion (X)
and the enantiomeric excess of the substrate (ees) and enan-
tiomeric excess of the product (eep) using the Equations30:
In our previous work,28,29 we compared five different
immobilization methods on chitosan beads and evaluated the
effects of glutaraldehyde on porosity of chitosan beads. Based
on the obtained experimental results, when glutaraldehyde
was used as an agent for both covalent bond and a cross‐linking
enzyme, better characteristics for immobilized enzyme than
other immobilization methods were shown. Subsequently,
covalent bonds with trapped free enzyme cross‐linked were
demonstrated as an appropriate method of immobilization for
chitosanbeads.Theaimofpresentstudywastoexaminethepri-
ority of immobilization of lipase on chitosan beads using phys-
ical adsorption and covalent bonds with entrapment of free
enzyme cross‐linked (termed activated chitosan beads). In
order to achieve high conversion and enantioselectivity of S‐
naproxen, theactivatedchitosanbeads wereused for thehydro-
lysis of naproxen methyl ester. Takac and Bakkal24
immobilized Candida rugosa lipase on Amberlite XAD7.
They determined the optimum reaction conditions in achieving
high activity and enantioselectivity for the hydrolysis of race-
mic naproxen methyl ester. Based on the obtained results,
Amberlite XAD7 was selected as the standard carrier for
enzyme immobilization in order to compare it with chitosan
CR−CS
CR þ CS
ees ¼
(1)
CS−CR
CR þ CS
eep ¼
X ¼
(2)
(3)
ees
ees þ eep
À
À
ÁÁ
Ln 1−X 1 þ eep
À
À
ÁÁ
E ¼
(4)
Ln 1−X 1−eep
where CR and CS in the ees equation denote concentration of
R‐enantiomer and S‐enantiomer of racemic naproxen methyl

GILANI ET AL.
3
ester, respectively. In addition, CR and CS in the eep equation
represent concentration of R‐naproxen and S‐ naproxen,
respectively.
In order to immobilize the enzyme on Amberlite XAD7,
the enzyme solution was prepared by dissolving 150 mg of
lipase enzyme in 500 mL buffer solution (50 mM pH 7 phos-
phate buffer solution). The solution was then brought in con-
tact with
5
g
Amberlite XAD7. Before enzyme
2.3 | Production of racemic naproxen methyl
immobilization, Amberlite XAD7 was washed with methanol
three times, and after that phosphate buffer solution was then
dried at room temperature overnight. The enzyme support
was shaken in an incubator shaker (Stuart, S1500 and UK)
at 30 °C, 150 rpm for 24 h. The pellets were separated from
solution by filtration and washed with buffer solution
(50 mM) at pH 7 and refrigerated at 4 °C.
ester
When a carboxylic acid (naproxen) is treated with alcohol
(methanol) and acid (H2SO4) as catalyst, ester (naproxen
methyl ester) is formed along with water. This reaction is
slow and reversible. In these cases, it may be necessary to
heat the reaction mixture under reflux for some time to pro-
duce an equilibrium mixture. By removing water, this reac-
tion is shifted to irreversible reaction so it can be
continuous to the end of one reactant. The water can be sep-
arated from the mixture using chloroform to make a biphasic
mixture and also a modified Dean‐Stark apparatus within
reflux; it can return the organic solvent to the reaction mix-
ture (Supporting Figure A1). To produce racemic naproxen
methyl ester, 3 g racemic naproxen was dissolved in
100 mL methanol and 100 mL chloroform with addition of
1 mL H2SO4 as catalyst. The mixture was heated and
refluxed by the modified Dean‐Stark apparatus at 70 °C bath-
water within 3 h. After that, the solvent was removed and pH
was adjusted to 9. The obtained precipitate was then washed
with 1 M NaHCO3 solution and distilled water. After drying,
racemic naproxen methyl ester powder was achieved with
yield of ~96% based on initial racemic naproxen. The synthe-
sized naproxen methyl ester was identified with FT‐IR. This
spectrum was compared to the commercial racemic naproxen
spectrum. All of the specific functional groups of FT‐IR
spectra are summarized in Table A1. Based on the obtained
results, the peaks of carboxylic acid in FT‐IR spectra of
naproxen (2967 and 1755 cm−1) were diminished in FT‐IR
spectra of naproxen methyl ester. In addition, the peak of
ester (1714 cm−1) appeared in the FT‐IR spectra of naproxen
methyl ester. The other functional groups were the same in
both of FT‐IR spectra.
2.5 | Hydrolysis reaction of racemic naproxen
methyl ester
Hydrolysis reactions were performed in three determined
batch experiments. Each experiment was carried out in a
serum bottle including of 5 mL organic solvent (25 mM race-
mic naproxen methyl ester solution), 5 mL buffer solution
(pH 7.0, 5 0 mM phosphate buffer solution), and one type
of carrier with enzyme immobilized on it. In these experi-
ments, 50 chitosan beads, 50 chitosan beads activated by glu-
taraldehyde, and 1 g of wet Amberlite XAD7 were
determined as a fixed value of each type of carrier. The reac-
tions were accomplished in an incubator shaker (Stuart,
S1500 and UK) at 35 °C, 150 rpm for 24 h. At the end of
reaction, 5 mL methanol was added to each reaction vessel
to stop the reaction and extraction of products. In order to
determine the conversion and enantioselectivity, samples
were drawn from the reaction mixture and analyzed by
HPLC.
3 | RESULTS AND DISCUSSION
3.1 | Effect of volumetric ratio of two phases
It has been recognized that the aqueous phase is crucial for
enzymatic reactions. In fact, the aqueous phase could be
improved by maintaining the active conformation of proteins
and enzyme dynamics.31 Therefore, it is reasonable that the
current study on the lipase hydrolysis reaction in organic sol-
vents considers the effects of volumetric ratio of the aqueous
phase to organic solvent. In our studies, the volumetric ratio
is referred to as the volumetric ratio of the aqueous buffer
solution to the organic solvent. Various volumetric ratios,
from 0.5:1, 1:1, 1.5:1, 2.5:1, 5:1, and 50:1 (v/v) were tested.
Organic solvent (isooctane) (5 mL) was added to buffer solu-
tion at values of 2.5, 5, 7.5, 12.5, 25, and 250 mL. Then the
mixtures were incubated in an incubator shaker at 35 °C,
150 rpm for 24 h. At the end of the reaction, the concentra-
tion of each enantiomer for naproxen and naproxen methyl
ester in the reaction mixture was assayed by HPLC analysis.
2.4 | Enzyme immobilization on support
Chitosan beads were prepared based on a procedure
described in the literature.28,29 In addition, enzyme was
immobilized on a chitosan support by physical adsorption
and covalent bond with trapped free enzyme cross‐linked
methods based on a procedure described in our previous
work.28
The specific enzyme activity was measured by colorimet-
ric method using p‐nitrophenyl palmitate (p‐NPP) as sub-
strate.28 The specific enzyme activity of chitosan beads,
activated chitosan beads by glutaraldehyde, and Amberlite
XAD7 was determined to be 120.7, 136.2, and 118.5 (U/g),
respectively.

4
GILANI ET AL.
Figure 1 (A and B) presents the conversion and enantioselectivity
of immobilized lipase on chitosan beads, activated chitosan
beads, and Amberlite XAD7 using different volumetric ratios
of aqueous buffer to organic solvent. By increasing the aqueous
buffer to organic solvent ratio until 1.5:1, both conversion and
enantioselectivity were increased for all supports. By increasing
the aqueous buffer solution more than 1.5 caused an intense
decrease in conversion and enantioselectivity for all carriers.
The reason for that may be immobilized enzyme was far from
the interface; therefore the specified enzyme could not
completely participate in the hydrolysis reaction. Therefore,
the volumetric ratio of aqueous phase to organic solvent at
1.5:1 for the rest of experiments was selected.
solvent (water‐miscible/immiscible). The kind of organic sol-
vent and reaction conditions are influential parameters on the
activity of the enzyme.33 In this multistep process, use of
immobilized enzyme can cause more complexity and diffi-
culties due to limiting substrate accessibility to the
immobilized enzyme. Consequently, finding new proper pro-
cedures is essentially required to ease the interfacial reaction
that may increase the activity and enantioselectivity of
immobilized lipase enzyme.
Therefore, three water‐miscible solvents (ethanol, ace-
tone, and 2‐ethoxyethanol), and three water‐immiscible sol-
vents (isooctane, cyclohexane, and n‐heptane) were selected
as organic solvents. The experiments were carried out with
5 mL organic solvent, 7.5 mL buffer solution, and determined
conditions as mentioned in a previous section. Table 1 sum-
marizes the effect of various organic solvents on the conver-
sion and enantioselectivity of immobilized enzyme into
chitosan beads, activated chitosan beads, and Amberlite
XAD7.
Based on the achieved data, isooctane had higher conver-
sion and enantioselectivity than other water‐immiscible sol-
vents for all supports. Since isooctane is more miscible in
water than n‐heptane and cyclohexane, naproxen methyl ester
could be able to reach the immobilized enzyme in the carrier.
On the other hand, lipase is a surface active enzyme which is
activated at the interface of two phases (hydrophobic vs.
hydrophilic); that is an important factor affecting the hydro-
lytic activity of lipase in an organic solvent. This is reason-
able because isooctane showed better conversion and
enantioselectivity than the water‐miscible solvent. Ethanol,
among all the examined organic solvents, had the lowest con-
version and enantioselectivity, probably due to inhibition of
solvent on lipase enzymatic activities. Consequently, isooc-
tane was selected as the desired organic solvent in the hydro-
lysis reaction.
3.2 | Effects of organic solvent
Due to low solubility of naproxen methyl ester in aqueous
media, it forms solid particles in suspension under the reac-
tion conditions. This may result in a suboptimal reaction rate
owing to poor substrate availability through low solubiliza-
tion rates. In this study, addition of the organic solvent,
cosolvent, and surfactant with the aim of increasing the solu-
bility of naproxen methyl ester, while retaining the enzyme
activity and enhancing substrate accessibility to immobilized
enzyme into the carrier, were investigated.
Based on the literature,5-7,32 the rate of hydrolysis reac-
tion is affected by the structure, polarity, and nature of the
However, enhancing the naproxen methyl ester accessi-
bility to immobilized enzyme plays a particularly important
role to improve conversion of the hydrolysis reaction. 2‐
Ethoxyethanol and acetone were examined as cosolvents for
isooctane in the reaction mixture because of their solubility
in both organic solvent and aqueous buffer. The experiments
were conducted with 5 mL organic solvent consisting of iso-
octane and cosolvent in a ratio of 1:1(v/v) and 7.5 mL buffer
solution.
Table 2 summarizes the effect of the cosolvent on conver-
sion and enantioselectivity. It can be concluded that 2‐
ethoxyethanol could be suitable as a cosolvent in the reaction
mixtures due to demonstrated better results than acetone for
all carriers.
In order to optimize the value of 2‐ethoxyethanol in the
organic solvent, further experiments were considered using
different volumetric ratios of 1:9, 2:8, 3:7, 4:6, and 5:5 (v/
v) of 2‐ethoxyethanol/isooctane.
FIGURE 1 The effect of volume ratio of aqueous buffer to organic
solvent on conversion (A) and enantioselectivity (B) of immobilized
lipase on chitosan bead, activated chitosan bead, and Amberlite XAD7;
reaction conditions: pH 7, 35 °C, 24 h

GILANI ET AL.
5
TABLE 1 The effect of various organic solvents on the conversion and enantioselectivity of immobilized enzyme into chitosan beads, activated
chitosan beads, and Amberlite XAD7
Organic solvent
Amberlite XAD7
Chitosan beads
Chitosan beads activated by glutaraldehyde
Water
Immiscible
Solvent
X
E
X
E
X
E
Isooctane
0.29
0.16
0.17
78.34
20.79
23.26
0.33
0.09
0.10
93.58
23.08
26.77
0.36
0.13
0.14
127.45
21.28
23.96
N‐heptane
Cyclohexane
Water
Miscible
Solvent
Acetone
0.26
0.25
0.12
38.25
41.69
14.15
0.29
0.31
0.16
68.39
84.97
34.21
0.32
0.33
0.16
95.94
110.38
29.41
2‐ethoxyethanol
Ethanol
Reaction conditions: pH 7, 35 °C, 24 h.
TABLE 2 The effect of cosolvent on conversion and enantioselectivity of immobilized enzyme into chitosan beads, activated chitosan beads, and
Amberlite XAD7
Solvent
Carrier
Acetone + isooctane (1:1 V/V)
2‐Ethoxyethanol + isooctane (1:1 V/V)
X
E
X
E
Amberlite XAD7
0.32
0.34
0.36
86.43
96.75
121.42
0.34
0.37
0.40
91.62
Chitosan beads
107.15
138.84
Chitosan beads activated by glutaraldehyde
Reaction conditions: pH 7, 35 °C, 24 h.
Figure 2 (A and B) indicate that high conversion and
enantioselectivity for all carriers were defined using the vol-
umetric ratio of 3:7 (v/v) 2‐ethoxyethanol to isooctane.
Based on the obtained results, isooctane was chosen as
the organic solvent and 2‐ethoxyethanol was added as
cosolvent at the optimum ratio of 3:7 (v/v) in 5 mL organic
solvent in the reaction mixture.
it may cause the enzyme to be more hydrophobic or more
hydrophilic, or may alter the enzyme configuration by attrac-
tion and repulsion forces. Therefore, it has a significant effect
on enzyme performance. The effect of pH on the hydrolysis
of racemic naproxen methyl ester was investigated for the
aqueous phase pH values of 6 to 8 with phosphate buffer
Figure 3 (A and B). These findings suggest that changes of
pH have no significant influence on the conversion of
immobilized lipase on chitosan beads activated by glutaralde-
hyde; however, it shows a higher conversion (0.42) than two
other types of supports. In addition, the high
enantioselectivity was obtained at neutral pH for all types
of supports. Consequently, the optimum pH value of hydroly-
sis reaction of naproxen methyl ester is defined at neutral pH
(pH of 7).
3.3 | Effect of pH
The influence of pH on the conversion and enantioselectivity
in the hydrolysis of racemic naproxen methyl ester can be
described as follows: 1) the changes of pH may alter the ion-
ization of the lipase enzyme that has influenced the enzyme
activity and selectivity; 2) the enantioselectivity of lipase
enzyme may be affected by the conformational change with
pH. This conformational change can occur as a result of the
displacement of its polypeptide functional group, which
could facilitate the access of S‐enantiomer of the racemic
mixture to the active center of the enzyme.1-3,10,24 The ions
of buffer solutions could change the solubility of the enzyme;
3.4 | Effect of temperature
The effect of temperature (25, 30, 35, and 40 °C) on the
hydrolysis of racemic naproxen methyl ester using lipase
immobilized on chitosan beads, chitosan beads activated by

6
GILANI ET AL.
FIGURE
3
The effect of pH on the conversion (A) and
enantioselectivity (B) of hydrolysis of racemic naproxen methyl ester;
reaction conditions: 35 °C, 24 h
FIGURE 2 The optimum ratio of 2‐ethoxyethanol to isooctane
toward conversion (A) and enantioselectivity (B) of immobilized
lipase on chitosan beads, activated chitosan beads, and Amberlite
XAD7; reaction conditions: pH 7, 35 °C, 24 h
glutaraldehyde, and Amberlite XAD7 was investigated. The
pH value of the aqueous phase of the hydrolysis medium
was neutral. An increase in temperature increased both the
conversion and enantioselectivity; however, a decrease was
observed at 40 °C Figure 4 (A and B). The highest conver-
sion and enantiomeric values for chitosan beads activated
by glutaraldehyde obtained at 35 °C were 0.42, and 183,
respectively.
3.5 | Time courses of the hydrolysis reaction
Figure 5 (A and B) shows the conversion and enantioselectivity
of immobilized lipase on support for the course of hydrolysis
of racemic naproxen methyl ester. The reaction was carried
out at the following conditions: pH of 7 aqueous phase, tem-
perature of 35 °C, volumetric ratio of aqueous phase to
organic phase 3:2; organic phase of 2‐ethoxyethanol/isooc-
tane in a ratio of 3:7 (v/v). The experiments were continued
for 48 h to complete the hydrolysis reaction. Because of the
fast reaction rate at the beginning of the experiment, every
3 h samples were collected until 12 h; after that sampling
was continued at intervals of 24 and 48 h. These obtained data
suggested that conversion of the hydrolysis reaction during
12 h could reach to more than 90% of its maximum efficiency.
FIGURE 4 The effect of temperature on the conversion (A) and
enantioselectivity (B) of hydrolysis of racemic naproxen methyl ester;
reaction conditions: pH 7, 24 h

GILANI ET AL.
7
glutaraldehyde is higher than chitosan beads and Amberlite
XAD7. The reason is that the high porosity of chitosan beads
activated by glutaraldehyde reduced the mass transfer limita-
tion of accessibility of the enzyme to racemic naproxen
methyl ester so that the reaction rate increased.
3.6 | Effect of surfactants
Surfactants are widely used in a lipase assay to raise the inter-
facial area. Since the reaction rate is intensely limited by the
low solubility of reactants, various surfactants were examined
as additives for the enzymatic reaction to improve the
conversion.
Based on the literature, when an anionic surfactant for
lipase enzyme was used, the ionic interactions between the
surfactant and lipase induced an unfolding and denaturing
of the enzyme; that may be the cause of the inhibitory effect
on the enzyme.34 In addition, cationic surfactants, due to their
positive charge, affected the active site of the solubilized
lipase so that it could reduce the efficiency of the surface‐
activity of the lipase. In addition, an interaction between the
positive charge of the head group in cationic surfactant and
negative charge of lipase could change the 3D structure of
the lipase so that it caused a decrease in enzyme activity.
When a nonionic surfactant for the lipase enzyme was used,
it created hydrogen‐bonding and hydrophobic interactions,
which is affected by the structure and charge of the enzyme.
This interaction could induce a conformational change that
favored the enzyme catalytic properties; thus, it can enhance
the activity and enantioselectivity of the enzyme.35 Liu
et al.35 used Tween‐80 as a nonionic surfactant for lipase
enzyme in hydrolysis of ketoprofen ester. They enhanced
the lipase enantioselectivity; as a result, it plays a particularly
important role in the enzymatic resolution. In addition, they
obtained nearly optically pure S‐ketoprofen, 96.4%. Their
findings suggest that Tween‐80 as an emulsifier provided a
stable substrate emulsion, which makes a great interfacial
area. In addition, it is an activator of the lipase enzyme,
which can enhance the enantioselectivity of the lipase
enzyme.
The effect of Tween‐80 on the enantioselectivity of the
lipase catalyzed hydrolysis of naproxen methyl ester was
investigated. In that research, Tween‐80 was added to the
organic solvent of the reaction mixture in a ratio of 0.1,
0.5, and 1% (v/v). Figure 6 (A and B) describes the obtained
value of conversion and enantioselectivity of the hydrolysis
reaction in the presence of surfactant to compare it to the
absence of the surfactant. The obtained results showed that
in a concentration of 0.1% surfactant, the conversion
increased from 0.39 to 0.45, and the enantioselectivity was
enhanced from 129 to 185 for the activated chitosan bead
by glutaraldehyde. Similarly, such an increase in conversion
and enantioselectivity were observed with Amberlite XAD7
FIGURE 5 The effect of time courses on the conversion (A) and
enantioselectivity (B) of hydrolysis of racemic naproxen methyl ester;
reaction conditions: pH 7, 35 °C
When the reaction continued till 48 h, the conversion did not
significantly increase.
For the enantioselectivity of immobilized lipase, similar
trends were observed.
According to the literature, enantioselectivity is a func-
tion of conversion and eep (eq. 4). Based on the obtained
results, the E value increased (nearly 2‐fold) by increasing
conversion vs. reaction time; but the ee of the product is con-
stant value (eep ≥ 96) within the reaction time.30 In enzy-
matic resolution reactions, the conversion increases quickly
at the beginning of the reaction and then reaches a constant
value. According to the our previous research,28 immobilized
lipase on chitosan beads followed a Michaelis–Menten
kinetic model, when inhibition was absent. Because of a high
concentration of naproxen methyl ester at the beginning of
the reaction, conversion increased with a sharp slope, while
after 12 h, concentration of the reactant decreased. Therefore,
the mass transfer driving force for accessibility of enzyme to
naproxen methyl ester severely decreased. Consequently,
conversion reached a constant value. Similar results for con-
version and enantioselectivity vs. time were reported in other
research.24,30
Consequently, to complete the reaction for all types of
carriers the hydrolysis reaction of racemic naproxen methyl
ester proceeded until 24 h. In addition, the hydrolysis rate
of immobilized enzyme on activated chitosan beads by

8
GILANI ET AL.
3.7 | Inhibitory effect of methanol
Through the hydrolysis of racemic naproxen methyl ester,
methanol as an undesired product is produced. The inhibitory
effect of a methanol concentration on the hydrolysis activity
of immobilized lipase on all mentioned carriers was investi-
gated. These experiments were carried out by addition of
methanol at different concentrations of 1 to 12% (v/v) to the
reaction mixture containing racemic naproxen methyl ester
and immobilized lipase on supports. As shown in Figure 7
(A and B), the conversion and enantioselectivity of the hydro-
lysis reaction rapidly decreased with an increase in methanol
concentration. This indicates that the methanol generated can
inhibit the activity of the immobilized lipase. The lowest con-
version and enantioselectivity at 12% methanol concentration
for immobilized enzyme on Amberlite XAD7 were 0.04 and
12.5, respectively. The immobilized enzyme on activated chi-
tosan beads was more resistant to the methanol concentration
than chitosan beads and Amberlite. The use of glutaraldehyde
as a coupling agent and cross‐linker in the immobilization
method combined together can improve the enzyme
stability on the fluctuation of the reaction mixture
microenvironment.
However, the inhibitory effect of methanol could be
neglected due to the methanol produced in a low concentra-
tion of naproxen methyl ester is not at a high value; also,
immobilized enzyme at concentrations of 3% (v/v) had no
FIGURE 6 The effect of surfactant on the conversion (A) and
enantioselectivity (B) of hydrolysis of racemic naproxen methyl ester;
reaction conditions: pH 7, 35 °C, 24 h
and chitosan beads. In contrast, addition of a surfactant con-
centration did not raise the value of conversion and
enantioselectivity of the hydrolysis reaction, probably due
to the foaming caused as a barrier in the reaction mixture.
It should be noted that the enantioselectivity and conver-
sion of free enzyme were determined in the obtained opti-
mum condition (5 mL organic solvent consisting of 2‐
ethoxyethanol, isooctane (3:7 v/v), and Tween‐80 0.1% (v/
v); 7.5 mL phosphate buffer; pH: 7, 35 °C, 24 h). The
enantioselectivity and conversion of Candida rugosa lipase
were obtained at 191 and 0.475, respectively, which are
higher than the enantioselectivity and conversion of an equal
amount of immobilized enzyme. These results are consistent
with other research; according to the literature,36-40 immobi-
lization may reduce enzyme activity and enantioselectivity by
the presence of a diffusion limitation. However, choosing an
appropriate immobilization method and suitable carrier can
mitigate the diffusion limitation.
In current research, enantioselectivity and conversion of
chitosan beads activated by glutaraldehyde are closed to
enantioselectivity and conversion of free lipase enzyme while
its reusability is very important for economical usage of the
lipase. Therefore, the developed technique can be introduced
as a suitable and biocompatible carrier which can overcome
diffusion limitation.
FIGURE 7 The inhibitory effect of methanol on the conversion (A)
and enantioselectivity (B) of hydrolysis of racemic naproxen methyl
ester

GILANI ET AL.
9
significant reduction on conversion and enantioselectivity of
the hydrolysis reaction. However, when a high substrate con-
centration is used for mass production of (S)‐naproxen, other
ester forms of racemic naproxen were required to generate a
noninhibitory product or methanol could be removed from
the reaction mixture by implementation of membrane reactor
or other techniques.
glutaraldehyde. After 10 times, the reuse of chitosan beads
activated by glutaraldehyde, conversion and enantiomeric
ratio declined to 0.25 and 55, respectively. While after
10 cycle of reuse, conversion and enantiomeric ratio of
immobilized enzyme on chitosan beads and Amberlite
XAD7 reached nearly 0.05 and 20, respectively. The main
reason for a reduction in enzyme activity is probably caused
by the denaturation or thermal inactivation of enzyme after
the sequence use of enzyme in batch runs. In addition,
because of weak binding of enzyme to the carrier, the leakage
of enzyme from the support may have occurred.
3.8 | Reusability of the immobilized lipase
The reusability of immobilized lipase was considered by con-
secutive consumption of supports in the hydrolysis reaction
of racemic naproxen methyl ester. Experiments were carried
out at optimum conditions as follows: aqueous phase pH of
7, temperature of 35 °C, volumetric ratio of aqueous phase
to organic phase 3:2; organic phase consisting of 2‐
ethoxyethanol/isooctane in a ratio of 3:7 (v/v), Tween‐80
0.1% (v/v), and duration of 24 h. After 24 h, all carriers were
recovered by filtration, then washed with phosphate buffer
solution (pH 7) and were reused for the next batch
experiments with the same conditions for 10 batch runs.
The variations in conversion and enantiomeric ratio are pre-
sented in Figure 8 (A and B). The conversion of 0.45 and
enantiomeric ratio of 185 was achieved at the end of the first
4 | CONCLUSION
Racemic naproxen methyl ester was synthesized by modified
Dean‐Stark and applied in an enzymatic hydrolysis reaction.
The lipase enzyme was immobilized on chitosan beads, acti-
vated chitosan beads by glutaraldehyde, and Amberlite
XAD7 as standard carrier. The optimum reaction mixture
and conditions were investigated for all supports and experi-
mental data were compared. For increasing the solubility of
racemic naproxen methyl ester in the reaction mixture, vari-
ous organic solvents were examined. Finally, isooctane and
2‐ethoxyethanol were selected as the suitable organic solvent
and cosolvent, respectively. The optimum hydrolysis reaction
conditions were found to be 35 °C, pH 7, and 24 h. The effect
of the addition of surfactant was considered and it success-
fully enhanced the conversion and enantioselectivity of the
hydrolysis reaction. Based on experimental data, the opti-
mum organic phase including 2‐ethoxyethanol/isooctane in
a ratio of 3:7 (v/v), and Tween‐80 0.1% (v/v) were deter-
mined. The best result was in conversion and
enantioselectivity of the immobilized enzyme in all experi-
mental runs devoted to chitosan beads activated by glutaral-
dehyde. The findings suggest that chitosan beads activated
by glutaraldehyde were successfully applied in the enzymatic
hydrolysis reaction of racemic naproxen methyl ester.
24
h of operation for chitosan beads activated by
ACKNOWLEDGEMENTS
The authors acknowledge Biotechnology Research Center,
Noshirvani University of Technology, and Department of
Pharmacology and Physiology, School of Medicine, Babol
University of Medical Sciences, Iran, for the facilities pro-
vided to accomplish this work.
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