Resolution of (R,S)-ibuprofen catalyzed by immobilized Novozym40086 in organic phase

Article abstract of DOI:10.1002/chir.23070

The enantioselective esterification of ibuprofen catalyzed by Novozym40086 was successfully conducted in organic solvent. Removing-water reagent was added into the reaction mixture to remove water produced in the esterification. The effects of temperature, n-hexanol concentration, ibuprofen concentration, and loading of enzymes were investigated. Under the condition of equilibrium, the thermodynamic equilibrium constant (K) of 7.697 and enantioselectivity (E) of 8.512 were obtained. The esterification reaction achieved its equilibrium in approximately 30?hours with conversion of 56% and ee_S of 93.78%. The predicted values of X and ee_S were 67.90% and 95.60%, respectively. The experimental value is approximately equal to the theoretical value, which indicates the feasibility of ideal models.

Full text of DOI:10.1002/chir.23070

Received: 12 December 2018
DOI: 10.1002/chir.23070
Revised: 10 March 2019
Accepted: 14 March 2019
R E G U L A R A R T I C L E
Resolution of (R,S)‐ibuprofen catalyzed by immobilized
Novozym40086 in organic phase
Xin Yuan | Lujun Wang | Guangyong Liu | Guilin Dai | Kewen Tang
Department of Chemistry and Chemical
Abstract
Engineering, Hunan Institute of Science
and Technology, Yueyang, China
The enantioselective esterification of ibuprofen catalyzed by Novozym40086
was successfully conducted in organic solvent. Removing‐water reagent was
added into the reaction mixture to remove water produced in the esterification.
The effects of temperature, n‐hexanol concentration, ibuprofen concentration,
and loading of enzymes were investigated. Under the condition of equilibrium,
the thermodynamic equilibrium constant (K) of 7.697 and enantioselectivity
Correspondence
Kewen Tang, Department of Chemistry
and Chemical Engineering, Hunan
Institute of Science and Technology,
Yueyang 414006, Hunan, China.
Email: tangkewen@sina.com
(
E) of 8.512 were obtained. The esterification reaction achieved its equilibrium
Funding information
National Natural Science Foundation of
China, Grant/Award Number: 21676077
in approximately 30 hours with conversion of 56% and ee of 93.78%. The pre-
S
dicted values of X and ee were 67.90% and 95.60%, respectively. The experi-
S
mental value is approximately equal to the theoretical value, which indicates
the feasibility of ideal models.
KEYWORDS
enantioselectivity, enzymatic kinetic resolution, esterification, ibuprofen, immobilized
Novozym40086
1
| INTRODUCTION
developed to produce optically pure drugs to meet the
growing market demand.
6
The living system is a chiral environment. From chemical
molecular level, chirality is an inherent characteristic in
living system. The chiral environment has a significant
The methods of obtaining single‐enantiomer drugs
mainly contain asymmetric synthesis and racemic resolu-
tion. Asymmetric synthesis includes fermentation, chiral
1-3
7
effect on biologically active substances. A pair of enan-
tiomers possess identical physical and chemical proper-
ties including chemical activity, solubility, density,
melting, and boiling point, but they often exhibit marked
source synthesis, and asymmetric catalytic synthesis.
Although new impressive progress has been obtained in
asymmetric synthesis, the most common way in industry
to obtain optically pure compounds is still via chiral reso-
lution of racemic mixtures. A series of resolution methods
2
differences in biological activities. In many cases, one
8
-10
enantiomer shows desired physiological effect, while the
other shows no effect or is even harmful to human
have been developed, such as membrane separation,
11,12
chromatography,
enantioselective
liquid‐liquid
and enzymatic kinetic resolu-
Membrane separation is still one of the most
4
13-15
health. Additionally, taking in optically pure drugs can
extraction (ELLE),
16-28
reduce the dose, reduce the metabolic burden, and reduce
tion.
2,3,5
the side effects caused by the undesired enantiomer.
widely applied technologies in manufacture pharmaceuti-
cal. But it has some disadvantages, for example, low
transport rates, fouling of membrane, and instability over
Nowadays, the market of single enantiomer is increasing
and how to obtain single‐enantiomer has become a hot
research field. A variety of chiral technologies have been
4
long periods of time. Chromatography, a key technology
for chiral separation, plays a crucial role in all fields, such
as pharmaceuticals, fine chemicals, food, and beverage
Xin Yuan and Lujun Wang contributed equally to this work.
Chirality. 2019;1–12.
wileyonlinelibrary.com/journal/chir
© 2019 Wiley Periodicals, Inc.
1

2
YUAN ET AL.
industries. Chromatography also possesses some advan-
tages, such as rapid separation, wide application scope,
convenient operation, high precision, and efficiency.
TM IM. Novozyme 435 is CAL‐B immobilized onto a
macroporous acrylic polymer resin. Novozym 40086, as
a new commercial immobilized lipase from Rhizomucor
miehei immobilized on acrylic resin beads, was a novel
and efficient biocatalyst for the esterification. Lipozyme
TL IM is a 1,3 specific lipase originating from
Thermomyces lanuginosus and immobilized on a noncom-
pressible silica gel carrier. Lipozyme RM IM is a 1,3 spe-
cific lipase originating from Rhizomucor miehei and
immobilized on a resin carrier.
29
However, large quantities of solvents are required and
much money is invested in expensive stationary phase
1,30
and high‐voltage equipment.
ELLE is regarded as a
promising technology because it is easy to realize contin-
uous production, easy industrial amplification, simple
operation, low production cost, and wide application
7,30,31
range.
The key problem for large‐scale industrial
applications of chiral extraction is lacking chiral extract-
ant with high selectivity and versatility. These drawbacks
In this study, the enantioselective esterification of ibu-
profen catalyzed by different immobilized lipases was
successfully conducted in organic solvent. Novozym
40086 was selected as the catalyst with the highest
enantioselectivity obtained. Two idealized models were
established, and matlab was used to simulate the effect
of substrate concentrations on ee. There is a good agree-
ment between experimental data and predicted values.
29
limit the practical application of ELLE.
Enzymatic kinetic resolution is an appealing alterna-
3
2
tive for chiral separation.
transesterification,
It can be applied in
16-19
20-22
23-28
hydrolysis,
esterification,
etc. Among enzymes used in kinetic resolution, lipases
33,34
(triacyglycerol acylhydrolases) are the most popular,
which shows the combination of wide substrate specific-
ity and high regioselectivity and enantioselectivity. As
lipases act as catalysts, the catalytic mechanisms are gen-
erally recognized as the “interfacial activation” mecha-
2
| EXPERIMENTAL
35-37
2.1 | Enzymes and chemicals
nism.
Two different structural forms of lipases are
assumed as follows: the closed form and the open form.
The closed form is considered as an inactive form, in
which the active sites are secluded from the reaction
medium by a helical oligopeptide chain called “lid.” How-
ever, the lid is displaced and the active sites are exposed
to reaction medium in the open form. For example, when
a hydrophobic substrate is catalyzed, such as an aromatic
substance, only the open form interacts with sub-
Novozym 435 from Candida antarctica and lipozyme TL
IM from Thermomyces lanuginosus were obtained from
Novo Nordisk company (Beijing, China). Lipozyme RM
IM and from Rhizomucor miehei and Novozym40086
from Aspergillus oryzae were obtained from Novo Nordisk
company (Beijing, China). Other property of the enzymes
was listed in Table 1. (R,S)‐ibuprofen was obtained from
Shanghai Titan Scientific Co., Ltd. (Shanghai, China).
Solvents for chromatography were of high‐performance
liquid chromatography (HPLC) grade. All other reagents
used in this work were of analytical grade and obtained
from different commercial suppliers.
36,37
strate.
Enzyme catalytic reaction has been widely
used due to its high stereoselectivity, broad substrate
specificity, enantioselectivity, and reactivity. At the same
time, the enzymatic kinetic resolution has a high thermo-
stability and conformational stability in hydrophilic and
32
hydrophobic environments.
‐Arylpropionic acids (profens) are known as major
2
2.2 | Analytical methods
nonsteroidal anti‐inflammatory drugs, which are used in
the treatment of cephalgia, muscular strain, headache,
The concentrations of (R)‐ibuprofen and (S)‐ibuprofen
were analyzed by HPLC (waters e2695, Waters Corpora-
tion, USA). An ultraviolet (UV) detection wavelength
38-40
and rheumatoid arthritis.
This drug class has a chiral
carbon atom within the propionic acid moiety. Ibuprofen
41
is one of the most frequently used drugs. The pharma-
cological activity of ibuprofen is mainly shown by the
was set at 230 nm.
A
chiral RJ‐OH column
(150 mm × 4.6 mm i.d., 5 μm) was employed, and the col-
umn temperature was maintained at 30°C. The mobile
phase was made up of methanol and 0.6% (V/V) acetic
acid aqueous solution (pH = 3.00, adjusted with
triethylamine). The volume ratio was 20:80. The flow rate
(
S)‐enantiomer, which is 160‐fold more active than the
42
corresponding (R)‐enantiomer.
Therefore, obtaining
the optically pure (S)‐enantiomer is of great significance.
Since lipases can be easily recovered from the reaction
medium, it can be immobilized on different carriers.
Immobilization of enzymes can change their secondary
structure modifying their activities. In this work, four
immobilized lipases were investigated as catalysts, includ-
ing Novozym 435, Novozym 40086, lipozyme RM IM, and
−
1
of the mobile phase was set at 1.0 mL min . The injec-
tion volume was 10 μL. The retention time of (R)‐ibupro-
fen was less than that of (S)‐ibuprofen. This analytical
4
3
method was adjusted according to the reference. The
product of ibuprofen n‐hexyl ester was analyzed by HPLC

YUAN ET AL.
3
TABLE 1 The property of lipases used in this paper
Lipases
Source
Status
Optimum T and pH
Activity
a
Novozym 435
Novozym40086
Lipozyme RM
Lipozyme TL
Candida antarctica
Aspergillus oryzae
Immobilized
Immobilized
Immobilized
Immobilized
T = 30–60°C pH = 5–9
T = 30–50°C pH = 7–10
T = 50°C pH = 7
1000
b
275
b
Rhizomucor miehei
Thermomyces lanuginosus
250
b
T = 50–75°C pH = 6–8
250
a
plu/g = Propyl Laurate Unit.
b
IUN/g = Interesterification Unit.
(waters e2695, Waters Corporation, USA). A Diamonsil
3 | METHODOLOGY
C18 column (250 mm × 4.6 mm i.d., 5 μm) was employed,
and the column temperature was maintained at 30°C. An
UV detection wavelength was set at 225 nm. The mobile
phase was made up of acetonitrile and 0.5% (V/V) phos-
phoric acid aqueous solution. The volume ratio was
In this reversible reaction, the reverse reaction has a sig-
nificant impact on reaction equilibrium. The process is
very complex, in which influences of various factors are
hard to be investigated. In order to simplify the process,
this study assumes an ideal reaction system.
In ideal reaction system, the reaction is completely
unaffected by its reverse reaction when it does not reach
equilibrium. Therefore, the influence of reverse reaction
ban be ignored. When the reaction reaches equilibrium,
the reaction rate is zero and the ee reaches its maximum
value. For this ideal process, the modeling process is as
follows.
9
1
0:10. The flow rate of the mobile phase was set at
.0 mL min . The injection volume was 10 μL. The
−
1
retention time of ibuprofen was less than that of ibupro-
fen n‐hexyl ester.
2
.3 | Enzyme‐catalyzed esterification of
ibuprofen
3
.1 | Determination of enantiomeric
The enzyme‐catalyzed esterification reaction was carried
out in a 25‐mL reaction flask. A certain concentration of
excess (ee) and conversion (c)
(
R,S)‐ibuprofen and concentration of n‐hexanol were
The enantiomeric excess (ee ) indicates the purity of a
s
added to 2‐mL methyl tert‐butyl ether (MTBE), and then
lipase was added to initialize the reaction. The reaction
was maintained under an agitate speed of 400 rpm. The
schematic diagram is shown in Figure 1. When the reac-
tion was completed, the reaction flask was cooled in an
ice‐water bath for 10 s, and the immobilized enzyme
was separated by filtration. Adding 0.5‐mL filtrate to a
single enantiomer in reaction substrate (Equation 1)
½
Sꢀ − ½Rꢀ:
S
ee ¼
(1)
½
Sꢀ þ ½Rꢀ
Conversion (X) of (R,S)‐ibuprofen was calculated as
follows:
1
0‐mL centrifuge tube and diluting the sample with
MTBE, the enantiomeric excess of substrate was deter-
mined by HPLC. The sample was diluted 10‐fold with
acetonitrile, and the products were analyzed by HPLC.
All experiments were repeated three times under identi-
cal conditions, and the precision level of the replicated
values was within ±3%.
½
Sꢀ þ ½Rꢀ ;
X ¼
(2)
½Sꢀ þ ½Rꢀ
0
0
where [S] and [R] are the substrate concentrations of
(S)‐ibuprofen and (R)‐ibuprofen after the reaction,
FIGURE 1 Biocatalysed esterification of (R,S)‐ibuprofen with n‐hexanol

4
YUAN ET AL.
respectively; [S] and [R] are the initial concentration of
decreasing the inverse‐reaction rate. Therefore, adding
excess concentration of alcohol or removing the water
can boost the process of esterification reaction to obtain
0
0
(
S)‐ibuprofen and (R)‐ibuprofen, respectively.
higher X and ees,max. The results can be proofed by fol-
lows description:
e
3
.2 | Establishment of relationship
between ees,max and equilibrium
conversion of fast‐reaction
The equilibrium conversion ratio (X ) of the enantio-
e
mers is related to the concentration of each reaction com-
ponent and the thermodynamic equilibrium constant (K)
in the reaction system. For esterification reaction, the fol-
lowing equation describes the reaction.
Assuming that the initial concentrations of (R) and (S)‐
enantiomers are equal, the reaction of (S)‐enantiomer is
faster than that of (R)‐enantiomer and ee can be defined
s
by the following equation:
R1COOH þ R2OH ⇌ H2O þ R1COOR2
XS − XR
A represents R COOH, B represents R OH, P repre-
eeS ¼
:
(3)
1
2
2
− XS − XR
sents H O, Q represents R COOR , and its thermody-
2
1
2
namic equilibrium constant (K) is given by following
equation.
Since it is unaffected by the reverse‐reaction, the
enantioselectivity (E) of the reaction is a constant that is
only related to the reaction system and can be calculate
by the following equation
CP⋅CQ
CA⋅CB
K ¼
:
(10)
The concentration of each component is an equilib-
rium concentration. C represents concentration of (R,
S)‐ibuprofen, C represents concentration of alcohol, C
ln½ð1 − XÞð1 − eeSÞꢀ
A
E ¼
(4)
ln½ð1 − XÞð1 þ ee Þꢀ
B
P
S
represents concentration of water, and C represents con-
Q
due to
centration of ester. The equilibrium conversion rate (Xe)
of (R,S)‐ibuprofen is defined by following equation.
ꢀ
X þ X ¼ 2X
XS − XR ¼ eeSð2 − XS − XRÞ ¼ 2eeSð1 − XÞ
S
R
:
(5)
CQ
Xe ¼
:
(11)
CA þ CQ
Equation 5 can be transformed into the following
equation
Combining Equations 10 and 11, X can be deduced
e
ꢀ
1
1
− X ¼ ð1 − XÞð1 − ee Þ
:
S
S
(6)
KCB
− XR ¼ ð1 − XÞð1 þ eeSÞ
Xe ¼
:
(12)
CP þ KCB
The relationship between XR and XS was obtained
according to Equations 4 and 6.
3
.3 | Ideal model of increase the alcohol
E
1
− XS ¼ ð1−XRÞ :
(7)
concentration
Combining Equations 3 and 7, Equation 8 can be
reduced
Combining eqs. (9) and (12), ees,max can be deduced.
2
2
eeS; max
¼
− 1
(13)
ee ¼
S
− 1:
(8)
ꢁ
ꢂ
E−1
E
E−1
E
CP
ð1−XSÞ þ 1
þ 1
CPþKCB
For an ideal system, ee will reach the maximum when
s
According to the mass balance, the concentrations of
X is equal to X .
S
e
components can be expressed as
2
eeS; max
¼
− 1
(9)
(
E−1
E
C ¼ CA;0⋅X
ð1−XeÞ þ 1
P
opt
;
(14)
CB ¼ CB;0‐CA;0⋅Xopt
From the Equation 10, it can be seen that the maxi-
mum value of ee can be obtained, which is only related
s
to the equilibrium conversion (X ). The ee
achieved by increasing the forward‐reaction rate or
can be
s,max
where CA,0 and CB,0 are the initial concentrations of the
enantiomeric acid and alcohol, respectively; Xopt is the
e

YUAN ET AL.
5
overall conversion rate of the enantiomers when the sys-
4.2 | Effect of temperature
tem ee reaches the maximum (ees,max). Combining Equa-
s
tions 13 and 14, the relationship between ees,max and Xopt
can be calculated as follows.
Temperature has an improtant impact on both enzyme
activity and enantioselectivity. The influence of tempera-
ture on conversion and enantiomeric excess of esterifica-
tion of (R,S)‐ibuprofen was tested with ranging from 40 to
80°C. As shown in Figure 2, the conversion of (R,S)‐ibu-
profen and ee increase with the increase of temperature
2
eeS; max ¼
− 1:
(15)
ꢃ
ꢄ
E−1
E
Xopt
þ 1
XoptþKðCB=CA−Xopt
Þ
(≤70°C), and then they decrease with further increase of
temperature (≥70°C). The maximum of conversion and
ee are reached at 70°C. E has no obvious change in this
S
3.4 | Ideal model of water concentration
range. Based on above results, 70°C is selected as the opti-
mum reaction temperature.
The concentration of water can be reduced by adding a
water removal agent in the reaction system. The
removal of water is performed by the hydration between
water removal agent and water. The amount of agent is
4.3 | Effect of (R,S)‐ibuprofen
concentration
excess, which can ensure the water concentration (C )
P
44
to be constant. Therefore, Equation 13 can be deduced
as follows.
Concentration of (R,S)‐ibuprofen has a significant influ-
ence on conversion, ee and E. Figure 3 shows the influ-
S
2
ence of different initial (R,S)‐ibuprofen concentration on
catalytic performance. As depicted in Figure 3, conver-
sion and ee are decreased with increasing (R,S)‐ibuprofen
concentration from 100 to 1200 mmol L . E is nearly
unchanged with a relative high value in the tested con-
centration of (R,S)‐ibuprofen. The results indicate that
the lower (R,S)‐ibuprofen concentration is, the higher
eeS; max ¼
− 1:
(16)
ꢃ
ꢄ
E−1
E
CP
þ 1
CPþKðCB‐CAXopt
Þ
−
1
4
4
| RESULTS AND DISCUSSION
.1 | Screening of lipases
conversion and ee are. However, the productivity should
S
also be considered, which will be low with low (R,S)‐ibu-
profen concentration. Based on these considerations, the
initial (R,S)‐ibuprofen concentration was selected at
Different lipases may show a large difference in the
catalytic efficiency of enantioselective esterification of
R,S)‐ibuprofen. Therefore, it is needed to obtain a lipase
600 mmol/L.
(
with good catalytic performance. The influences of four
commercially available lipases on enantioselective
esterification of (R,S)‐ibuprofen with n‐hexanol were
investigated, in which MTBE acted as organic solvent.
Table 2 displays the results of ee , X, and ee after
4.4 | Effect of n‐hexanol concentration
The estrification reaction catalyzed by enzyme is revers-
ible. To prompt the formation of product, excessive con-
centration of n‐hexanol is added. However, alcohols act
as a nucleophile and excessive n‐hexanol concentration
will affect the enzyme activity and the enantioseletivity.
As shown in Figure 4, the conversion of (R,S)‐ibuprofen
s
p
2
4 hours. Three lipases (lipase 435, 40086, and RM) show
excellent catalytic performance, while lipase TM shows
no catalytic performance. Compared with other lipases,
Novozym40086 shows the highest catalytic performance
and selectivity. Therefore, Novozym40086 was selected
as the catalyst.
and ee are decreased with increasing n‐hexanol concen-
s
tration from 450 to 2000 mmol/L. E has no obvious
TABLE 2 Results of esterification of (R,S)‐ibuprofen catalyzed by different lipases
Lipases
Source
ee
s
, %
X, %
p
ee , %
Novozym 435
Novozym 40086
Lipozyme RM
Lipozyme TL
Candida antarctica
Rhizomucor miehei
Rhizomucor miehei
Thermomyces lanuginosus
5.42
71.03
18.41
0
33.35
25.60
12.21
0
25.71
87.40
39.51
0
Note. Conditions: C
A
= 100 mmol/L, C
B
= 100 mmol/L, and T = 70°C.

6
YUAN ET AL.
S
FIGURE 2 Effects of reaction temperature on X, ee , and E. Conditions: 100 mmol/L C
A
, 100 mmol/L C
B
, 100 mg/mL Novozym40086, and
reaction time 1 hour
S B
FIGURE 3 Effects of (R,S)‐ibuprofen concentration on X, ee , and E. Conditions: 600 mmol/L C , 100 mg/mL Novozym40086,
temperature 70°C, and reaction time 1 hour
change with n‐hexanol concentration ranging from 450 to
4.5 | Effect of enzyme amount
8
00 mmol/L and then decreases with further increase of
n‐hexanol concentration (≥800 mmol/L). The results
indicate that high n‐hexanol concentration probably
inhibits the enzyme activity and the enantioseletivity.
Furthermore, high n‐hexanol concentration may increase
the occurrence of nonenantioselectivity reaction. There-
fore, the n‐hexanol concentration chose 800 mmol/L to
obtain higher X.
Lipase as a catalyst, increasing the amount of lipase can
accelerate the reaction. In order to obtain optimal
enzyme amount, the effect of enzyme amount on
enantioselective esterification of (R,S)‐ibuprofen was
investigated. As shown in Figure 5, the conversion and
45
ee are increased linearly with increasing the amount of
s
immobilized enzyme. The E is decreased slowly with

YUAN ET AL.
7
FIGURE 4 Effects of n‐hexanol concentration on X, ee
S
, and E. Conditions: 600 mmol/L C
A
, 100 mg/mL Novozym40086, temperature
7
0°C, and reaction time 1 hour
S A B
FIGURE 5 Effects of enzyme amount on X, ee , and E. Conditions: 600 mmol/L C , 2000 mmol/L C , 100 mg/mL Novozym40086,
temperature 70°C, and reaction time 3 hours
the increase of immobilized enzyme. This phenomenon
indicates that the increase of the amount of enzyme
shows no inhibitation of lipase catalytic activity. Using
immobilized enzyme can avoid the tendency of lipases
to give aggregates. Therefore, the amount of enzyme
can be selected based on actual requirements (such as
controlling the reaction rate and controlling the produc-
tion cost).
4.6 | Determination of thermodynamic
equilibrium constant (K) and
enantioselectivity (E)
4
6
Under specified solvent and temperature, K is constant
and E can be considered as a constant before the fast‐
reacting species attaining equilibrium. According to
Equations 5 and 16, K and E are important parameters

8
YUAN ET AL.
for determining ees,max, so the determination of them is
needed before the calculation of ees,max. When fast‐
reaction reached the thermodynamic equilibrium, the
gated. When the ee reaches the maximum, the conver-
s
sion of the fast‐reaction was up to the highest degree
and then was constant. At this time, K and E were calcu-
lated by the Equations 5 and 11, respectively. As shown
in Figure 6, it is also found that there is a rapid increase
of X , X , and ee at first, and then a slow increase of them
ee was up to the maximum (ee
s
). ee is a function
s
s,max
of conversion, and conversion is a function of time.
Therefore, the Novozym40086 catalysis esterification of
S
R
(
R,S)‐ibuprofen with different reaction time was investi-
happens after 6 hours. Therefore, the reaction can be
R S S A B
FIGURE 6 Effects of reaction time on X , X , ee , and E. Conditions: 100 mmol/L C , 100 mmol/L C , 100 mg/mL Novozym40086, and
temperature 70°C
R S S A B
FIGURE 7 Effects of reaction time on conversion X , X , and ee . Conditions: 600 mmol/L C , 800 mmol/L C , 100 mg/mL
Novozym40086, and temperature 70°C

YUAN ET AL.
9
regarded as reaching equilibrium at 6 h. K and E were
calculated by Equations 5 and 11, with values of 7.697
and 8.512, respectively.
and (16). The relevant parameters of the enzyme catalytic
reaction have been depicted in Table 3.
4.8 | Model optimization and validation
Combined with Equations 3, 11, and 16, matlab was used
to simulate the effect of substrate concentrations on eeS.
4
.7 | Calculation and experimental
verification
As shown in Figure 8, ee increases rapidly and then
S
shows no change when CB is more than 800 mmol/L.
The factors affecting X , X , and ee were optimized, such
R
S
S
The ee decreases with the increase of C , and it can keep
A
as temperature, Novozym40086 amount, and substrate
concentrations. Under these conditions, the influences
of reaction time on X , X , and ee were investigated.
a relative high value (≥95%). Taking the economy of n‐
hexanol and the productivity of (R,S)‐profen into consid-
R
S
−
1
eration, 800 mmol/L CB and 600 mmol L
CA are
From Figure 7, it can be found that X , X , and ee
R
S
selected to achieve high X and ee.
increase with reaction time. The X and ee reach the max-
S
imum at 36 hours. The ee is up to 93.78%, which is close
s
to the expected ee value 95.6% estimated by Equations 5
S
4.9 | Application
TABLE 3 The relevant parameters of the enzyme catalytic
reaction
The optimal reaction conditions were obtained according
to the above results, including 800 mmol/L n‐hexanol,
600 mmol/L (R,S)‐ibuprofen, 100 mg/mL Novozym40086,
Parameter
Value
7
0°C, agitation speed of 400 rpm, and 30 hours for reac-
Expected value of ees,max
95.6%
7.697
8.512
40
tion time. Under the optimal conditions, the experimen-
tal values of X and eeS are 59.90% and 93.78%,
respectively, and the predicted values of X and ee are
67.90% and 95.60%, respectively, which indicate that the
experimental values are well in agreement with the
model prediction. The enantiomeric excess of the sub-
strate was analyzed by HPLC (Figure 9). Figure 9A,B
K
E
S
C
C
C
P
/mmol/L
/mmol/L
/mmol/L
A
600
B
800
s A B
FIGURE 8 Dependence of predicted ee on C and C . Conditions: 100 mg/mL Novozym40086 and temperature 70°C

10
YUAN ET AL.
FIGURE 9 High‐performance liquid chromatography chromatograms of the ibuprofen enantiomers. A, Racemate of (R,S)‐ibuprofen. B,
The remaining (R,S)‐ibuprofen after esterification
shows the chromatograms of racemic (R,S)‐ibuprofen and
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How to cite this article: Yuan X, Wang L, Liu G,
Dai G, Tang K. Resolution of (R,S)‐ibuprofen
catalyzed by immobilized Novozym40086 in
organic phase. Chirality. 2019;1–12. https://doi.org/
10.1002/chir.23070
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