pH memory of immobilized lipase for (±)-menthol resolution in ionic liquid

Article abstract of DOI:10.1021/jf073067f

Magnetic DEAE-GMA-EDMA microspheres were prepared via suspension polymerization and used for the immobilization of Candida rugosa lipase by ion exchange. The effect of pH values on the immobilization of lipase was investigated. Resolution of (±)-menthol in the hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate was performed by immobilized lipase-catalyzed enantioselective esterification with propionic anhydride as acyl donor. The effects of pH condition at lipase immobilization on the conversion and enantioselectivity were investigated. As a result, pH memory of the immobilized lipase for catalyzing (±)-menthol resolution in the ionic liquid was observed. Better conversion and the best enantioselectivity were obtained with the immobilized lipase prepared at pH 5.0. Under the condition, (-)-menthyl propionate with enantiomeric excess of >90% was obtained. Moreover, the enantioselectivity of the immobilized lipase decreased gradually with increasing pH value.

Full text of DOI:10.1021/jf073067f

2388 J. Agric. Food Chem. 2008, 56, 2388–2391
pH Memory of Immobilized Lipase for (()-Menthol
Resolution in Ionic Liquid
MENG-YUAN REN, SHU BAI, DONG-HAO ZHANG, AND YAN SUN*
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin
University, Tianjin 300072, China
Magnetic DEAE-GMA-EDMA microspheres were prepared via suspension polymerization and used
for the immobilization of Candida rugosa lipase by ion exchange. The effect of pH values on the
immobilization of lipase was investigated. Resolution of (()-menthol in the hydrophobic ionic liquid
1-butyl-3-methylimidazolium hexafluorophosphate was performed by immobilized lipase-catalyzed
enantioselective esterification with propionic anhydride as acyl donor. The effects of pH condition at
lipase immobilization on the conversion and enantioselectivity were investigated. As a result, pH
memory of the immobilized lipase for catalyzing (()-menthol resolution in the ionic liquid was observed.
Better conversion and the best enantioselectivity were obtained with the immobilized lipase prepared
at pH 5.0. Under the condition, (-)-menthyl propionate with enantiomeric excess of >90% was
obtained. Moreover, the enantioselectivity of the immobilized lipase decreased gradually with increasing
pH value.
KEYWORDS: pH memory; ionic liquid; immobilized lipase
INTRODUCTION
found that adding a micro amount of phosphate buffer of pH
7.0 could increase the conversion yield of esterification and
verified that lipase had pH memory in organic solvents. Up to
now, studies of lipase’s pH memory are still focused on the
reaction system in organic solvents. Whether immobilized lipase
also has pH memory in ionic liquids and how it takes effect on
the activity and enantioselectivity of lipase have not been
reported in the literature.
As an alternative to traditional organic solvents, ionic liquids,
a new class of green solvents, have attracted many investigations
for applications in biocatalysis and biotransformations. Recent
research findings have shown that many enzymes keep high
activity in ionic liquids, even in an almost anhydrous
environment (1–3). In nonaqueous enzymatic catalysis system,
however, the macro-pH of the reaction media cannot be
measured, nor can the micro-pH of essential water solution
around the enzyme molecules. Therefore, control of the pH value
of the reaction media in nonaqueous environments seems to be
impossible. Zaks and Klibanov (4) dissolved lipase in buffers
of different pH values, then lyophilized it, and thus prepared
the lipase at different initial pH values. The lipase was then
employed to catalyze the hydrolysis of tributyrin in organic
solvents. They found that the hydrolysis activity of such lipase
with “pH history” had noticeable correlation with the pH of
the initial buffers. They introduced the concept for the first time
that lipase had “pH memory” in organic solvents. Further
research (5) on immobilized lipase was made, and the results
demonstrated that immobilized lipase also had pH memory; that
is, lipase would remember the pH of the final aqueous
environment during its immobilization. Therefore, immobilizing
lipase at the optimal pH value will benefit its activity in
nonaqueous systems. Manohar et al. (6) studied porcine pancreas
lipase-catalyzed esterification of anthranilic acid with methanol
in organic solvents by an artificial neural network analysis. They
In this work, magnetic GMA-EDMA microspheres of about
5 µm were prepared by suspension polymerization and then
modified to an anion exchanger with DEAE-Cl. Candida
rugosa lipase was adsorbed on the magnetic carrier by ion
exchange in buffers of different pH values. After lyophilization,
the immobilized lipase would retain the pH history of the buffers
and was used to esterify and resolve (()-menthol in the
hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexaflu-
orophosphate ([Bmim]PF6). Here, the enzyme-catalyzed esteri-
fication of (()-menthol in ionic liquid with propionic anhydride
as acyl donor was measured. Especially, it is necessary to
investigate the effect of pH on the lipase-catalyzed enantiose-
lective esterification of (()-menthol, which was not considered
by previous researchers (7–9). Comparison of the activity and
enantioselectivity between the free and immobilized lipase in
ionic liquid was also performed. The effects of pH value on
the activity of the two types of lipases and the capability of pH
memory of the immobilized lipase in ionic liquid were studied.
Thus, we can control the micro-pH of the essential water
solution around the enzyme molecules in ionic liquids and
further optimize the enzyme activity for enantioselectively
esterifying (()-menthol.
* Corresponding author (telephone +86 22 27404981; fax +86 22
27406590; e-mail ysun@tju.edu.cn).
10.1021/jf073067f CCC: $40.75  2008 American Chemical Society
Published on Web 03/14/2008

pH Memory of Immobilized Lipase in Ionic Liquid
J. Agric. Food Chem., Vol. 56, No. 7, 2008 2389
MATERIALS AND METHODS
Materials. (()-Menthol, (-)-menthol, propionic anhydride, diethy-
laminoehtyl chloride (DEAE-Cl), Coomassie brilliant blue G-250,
bovine serum albumin (BSA), and C. rugosa lipase (CRL, type VII)
were obtained from Sigma (St. Louis, MO). Ethylene glycol dimethacry-
late (EDMA) was obtained from Fluka (Buchs, Switzerland). FeSO4,
FeCl3, glycidyl methacrylate (GMA), (2,2)-azoisobisbutyronitrile (AIBN),
polyvinyl alcohol (PVA), dodecyl alcohol, sodium dodecyl sulfate
(SDS), and all other chemicals were of analytical grade and obtained
from local sources.
Preparation of Magnetic DEAE-GMA-EDMA Microspheres.
Fe3O4 magnetic fluid of nanometer was prepared via chemical copre-
cipitation process (10). Magnetic poly(GMA-EDMA) microspheres
were prepared via suspension polymerization (11). The organic phase
contained magnetic fluid (1.00 g, wet weight), GMA (1.50 g), EDMA
(2.10 g), dodecyl alcohol (0.40 g), and 0.30 g of AIBN. The aqueous
continuous phase was composed of SDS (0.80 g) in 300 mL of 1%
PVA solution. The polymerization reactor, a three-necked flack
equipped with a mechanical stirrer, was hatched in a water bath and
heated to 80 °C. The stirring speed was set to 100 rpm After 5 h of
reaction, the acquired composite magnetic microspheres were collected
by a permanent magnet and then were sequentially washed with
deionized water, ethanol, and acetone. The product was reacted with
NaBH4 to convert the epoxy groups on the microspheres’ surface into
hydroxyl groups. The microspheres then were modified with DEAE-
Cl. The thus obtained magnetic DEAE-GMA-EDMA microspheres had
a mean diameter of 5 µm and could be used as the carrier of lipase
immobilization.
Figure 1. Kinetics of the ion exchange immobilization of Candida rugosa
lipase at different pH values (9, pH 5.0; [, pH 6.0; b, pH 7.0; 2, pH
8.0).
detector, and a CYCLOSIL-B capillary column (0.25 µm film thickness,
30 m length, 0.25 mm i.d.). The injector and detector were set at 200
and 250 °C, respectively, and nitrogen was used as the carrier gas.
The oven temperature was kept at 90 °C for 10 min, programmed to
increase from 90 to 150 °C at 2 °C/min, then increased to 165 at 5 °C
/min, and finally kept at 165 °C for 5 min. Chromatographic data were
acquired and analyzed using the Agilent Chemical Station. The retention
times were 30.5 and 31.5 min for (-)- and (+)-menthyl propionate,
respectively, and 26.3 min for (()-menthol.
Immobilization of Lipase onto Magnetic DEAE-GMA-EDMA
Microspheres. Magnetic DEAE-GMA-EDMA microspheres of the
same dry weight were washed with buffers (50 mmol/L) of different
pH values and then suspended in the corresponding buffers, respectively.
C. rugosa lipase powder was added to the suspension with a dry carrier-
to-enzyme weight ratio of 1:1. The reaction was carried out at 25 °C
with shaking at 150-170 rpm. At definite time intervals, the micro-
spheres with immobilized lipase were collected by permanent magnet
and washed twice by the same buffer solution, respectively. Then they
were lyophilized and stored at -20 °C for future use.
The amount of lipase protein immobilized on the carrier was
evaluated by determining the soluble protein content in the remaining
solution according to the Bradford method (12). Activities of the
immobilized lipase were determined with the method of olive oil
hydrolysis (13). One unit of enzyme activity was defined as the amount
of lipase that liberates 1 µmol of fatty acids per minute under the assay
condition. The specific activity of immobilized lipase was calculated
as the ratio of enzyme activity to the total amount of immobilized
enzyme. Experiments were conducted in triplicate. The mean values
are presented, and standard deviations are given as error bars in all
figures (see below).
Preparation of [Bmim]PF6. The hydrophobic ionic liquid
[Bmim]PF6 was prepared according to the procedure described by
Huddleston (14). The ionic liquid was lyophilized for 24 h, and the
water activity was 0.07 as assayed by Hygrolab (Rotronic,
Switzerland).
Esterification of Menthol. A typical experiment was carried out as
described in a previously published paper (15): 1.0 mmol of (()-
menthol and a certain amount of immobilized CRL (100 units) were
added to 3 mL of the ionic liquid in a 10 mL screw-capped vial. The
reaction was started after the addition of 1.0 mmol of propionic
anhydride and run by shaking at 200 rpm at a temperature of 30 °C for
24 h. At different time intervals, aliquots were taken and analyzed by
gas chromatography (GC) after extraction and dilution with n-hexane.
Immobilized lipase was separated by permanent magnet when the
reaction finished and could be reused.
RESULTS AND DISCUSSION
Effect of pH Value on the Adsorption of Immobilized
Lipase. The effect of pH value on the adsorption of free lipase
was investigated by immobilizing lipase in buffers of different
pH. Figure 1 shows the adsorption kinetics in buffers of pH
5.0, 6.0, 7.0, and 8.0. It can be seen that pH values affect the
adsorption of lipase greatly. The amount of lipase immobilized
on the carrier at pH 8.0 was about twice that at pH 5.0. On the
other hand, the time point when the highest adsorption amount
is reached varies with pH value. For example, at pH 8.0, the
loading capacity reaches the maximum at 120 min, whereas at
pH 5.0 the time is 60 min.
Some papers (17–19) have reported that although commercial
C. rugosa lipase (type VII, Sigma) had been rid of most impurity
proteins, it was still composed of some isoenzymes presenting
different catalysis behaviors, such as enantioselectivity. In many
resolution reactions (17–19), the enantioselectivity of these
isoenzymes was distinctive, even opposite. Two components
in commercial CRL were separated by ion exchange chroma-
tography, namely, CRLA and CRLB. As analyzed on poly-
acrylamide gel electrophoresis under denaturing conditions
(SDS-PAGE), CRLA and CRLB are both in the same molecular
weight range of 62-64 kDa. On isoelectric focusing, CRLA
showed a single band corresponding to an isoelectric point (pI)
of 5.6 and CRLB was resolved in two bands having pI values
around 4.2 (17). At pH 5.0, only CRLB is negatively charged
and can be adsorbed onto DEAE-functionalized anion ex-
changer. Thus, under this condition, the carrier adsorbed only
CRLB and the lipase loaded on the carrier is the lowest. As the
pH is increased, more proteins are negatively charged, so the
adsorption capacity increases correspondingly. These adsorption
curves also give the optimal times of enzyme loading at different
pH values. The optimum immobilization time is shortest at pH
5.0 because less protein is immobilized at this condition.
Effect of pH on the Activity of Immobilized Lipase. The
activity and specific activity of the immobilized lipases prepared
at different pH values were investigated to examine the effect
The enantiomeric excess (ee%) and the conversion of menthol (c)
were based on the GC analyses and calculated by the equations as used
previously (16).
GC Analysis. The GC analysis was performed as described
previously (15) with an Agilent 6890N (Agilent Technologies, Wilm-
ington, DE) equipped with a splitless/split injector, a flame ionization

2390 J. Agric. Food Chem., Vol. 56, No. 7, 2008
Ren et al.
Figure 2. Effect of pH on the specific activity (solid bars) and activity
(open bars) of the immobilized lipase.
Figure 3. Effect of immobilization pH on the conversion of menthol and
enantiomeric excess. Menthol conversion: 9, pH 5.0; b, pH 7.0; 2, pH
8.0. Enantiomeric excess (ee%): 0, pH 5.0; O, pH 7.0; 4, pH 8.0.
of pH on the biocatalysis activity. The results are shown in
Figure 2. It can be seen from the figure that the pH value shows
dramatic impacts on activity and specific activity of the
immobilized lipase, especially on the latter. The highest activity,
452 units/g of carrier, appears at pH 7.0. At pH 5.0 and 8.0,
the activity was relatively lower (about 320 units/g of carrier).
As for the specific activity, the highest value (1240.31 units/g
of protein) appears at pH 5.0, whereas the lowest one, 614.20
units/g of protein, appears at pH 8.0. The results demonstrate
that at different pH environments, higher enzyme adsorption
does not mean higher catalytic activity (see also Figure 1). Such
results might be attributed to the fact that pH 8.0 is much higher
than the pI of the lipase, resulting in the change of the secondary
structure and tertiary structure as well as the conformation of
active center in such a basic environment. Thus, the activity
and specific activity of the lipase decrease at pH 8.0. On the
contrary, in an acidic environment of pH 5.0, although the
enzyme loading capacity is the lowest, selective adsorption of
CRLB and the intact natural structure of lipase result in the
highest specific activity. These results are in agreement with
the finding that CRL has a higher activity in neutral or weak
acid environment (20). On the other hand, most impurity
proteins in the commercial CRL have pI > 5.0, so the
isoenzymes and impurities are most adsorbed on the carrier at
pH 6.0-8.0, which decreases the specific activity of the
immobilized lipase.
pH Memory of the Immobilized Lipase in Ionic Liquid.
Resolution of (()-menthol was carried out in the hydrophobic
ionic liquid [Bmim]PF6 by employing the immobilized lipase
with different pH histories (pH 5.0, 7.0, and 8.0). The results
are shown in Figure 3. The results indicate that the conversion
of menthol is highest at the neutral condition (pH 7.0), whereas
it is lowest at the basic condition (pH 8.0). This is consistent
with the results discussed above. Moreover, pH also presents a
significant effect on the enantiomeric excess. Lipase im-
mobilized at pH 5.0 gives the best result of resolving the
enantiomers. The enantiomeric excess decreased drastically with
increasing pH at the reaction time of 24 h. These results
demonstrate that the lipase has a pH memory effect in ionic
liquid.
value, as illustrated in this work, which is coincident with that
reported previously (22).
In many resolution reactions, the enantioselectivity of isoen-
zymes of lipase is greatly different, or even opposite (18, 19).
In our research, the immobilized lipase with pH 5.0 history is
mostly CRLB, which conformation may be more suitable for
the stereoselective resolution of certain menthol enantiomer in
ionic liquid. Given that the mechanism and regularity of the
medium’s impact on lipase enantioselectivity are unclear, further
research is necessary.
The effect of pH on the ionization of lipase determines its
conformation (13). At a certain pH condition, the ionic groups
around the active center of the lipase molecule achieve the
optimum ionic state for lipase-catalyzed reactions, which is
essential to its activity and enantioselectivity (23). In traditional
aqueous system, the pH value of lipase-catalyzed hydrolysis
reaction is adjusted with bulk buffers. In a nonaqueous reaction
system, however, water activity has a significant influence on
the activity, and water content must be under rigorous control.
Hence, it is impossible to control the pH of the reaction by
adding buffers. The hydrophobic ionic liquid [Bmim]PF6, which
is a liquid salt at vicinal room temperature (from -30 to
50 °C), is also sensitive to the water activity as a medium of
lipase-catalyzed reaction. The C. rugosa lipase has activity and
enantioselectivity only at very low water content (24). Therefore,
we made immobilized lipase with pH history during the
preparation procedure. Such an immobilized lipase with pH
history was used to resolve (()-menthol in the ionic liquid
[Bmim]PF6 (water activity aw ) 0.07). Experimental results
verified that essential water around the lipase molecules
remained at the immobilization pH when it was used in the
ionic liquid reaction system.
Zaks and Klibanov (4, 5) had reported that free and
immobilized lipases both had pH memory effects in organic
solvents after lyophilization. Here, we found that in a novel
green nonaqueous solvent–ionic liquid, immobilized CRL also
had pH memory after lyophilization, and a new method was
supplied to prepare immobilized lipase with pH memory. Via
a simple and easily performed selective adsorption, we are able
to combine the immobilization with the purification. This
method is suitable to separate isoenzymes of slight structure
difference and improves the catalytic activity of enzymes.
Especially, the control and adjustment of pH were realized in
biocatalysis with ionic liquid as a reaction medium, which is
advantageous in the production of some food additives via
enzyme technology.
Previous studies (20, 21) have suggested the optimal pH
values at which CRL displayed the best activity of esterification
and transesterification in nonaqueous media. Although the
optimum pH value herein is not identical with that of previous
work (17, 18) because of the different reaction system and
method of assay, the results in this research agree with the
widely recognized phenomenon that CRL shows higher activity
in neutral and weak acid environments. Furthermore, the
enantioselectivity of lipase was reduced with increasing pH

pH Memory of Immobilized Lipase in Ionic Liquid
J. Agric. Food Chem., Vol. 56, No. 7, 2008 2391
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