Microreactor with mesoporous silica support layer for lipase catalyzed enantioselective transesterification

Article abstract of DOI:10.1039/b917374a

Lipase PS was immobilized in mesoporous silica (MPS) thin films inside a borosilicate tube microreactor for use in the enantioselective transesterification of vinyl acetate with (±)-1-phenylethanol. The immobilization was tested for 3D cubic and 2D hexagonal MPS thin films inside microreactors with and without hydrophobic treatment. The hydrophobic treatment enhanced the adsorption amount and the lipase activity for both 3D cubic and 2D hexagonal films. Of these treated films, the 3D cubic structure film exhibited the highest yield (64%) with an enantioselectivity higher than 99% in a continuous flow experiment. The activity of the immobilized lipase PS was well maintained for 36-hour continuous operation. A Ping-Pong Bi Bi kinetic model was employed to interpret the activity of the immobilized lipase PS. The ratios of the maximum velocity to the Ping-Pong constant (i.e., specificity constants) were measured for lipase PS immobilized inside the microreactor and for native lipase PS. The value inside the microreactor was 800 times greater than that of the native lipase PS.

Full text of DOI:10.1039/b917374a

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www.rsc.org/greenchem | Green Chemistry
Microreactor with mesoporous silica support layer for lipase catalyzed
enantioselective transesterification†
Sho Kataoka, Yasutaka Takeuchi, Atsuhiro Harada, Mitsuhiko Yamada and Akira Endo*
Received 22nd August 2009, Accepted 21st November 2009
First published as an Advance Article on the web 18th January 2010
DOI: 10.1039/b917374a
Lipase PS was immobilized in mesoporous silica (MPS) thin films inside a borosilicate tube
microreactor for use in the enantioselective transesterification of vinyl acetate with
(
±)-1-phenylethanol. The immobilization was tested for 3D cubic and 2D hexagonal MPS thin
films inside microreactors with and without hydrophobic treatment. The hydrophobic treatment
enhanced the adsorption amount and the lipase activity for both 3D cubic and 2D hexagonal
films. Of these treated films, the 3D cubic structure film exhibited the highest yield (64%) with an
enantioselectivity higher than 99% in a continuous flow experiment. The activity of the
immobilized lipase PS was well maintained for 36-hour continuous operation. A Ping-Pong Bi Bi
kinetic model was employed to interpret the activity of the immobilized lipase PS. The ratios of
the maximum velocity to the Ping-Pong constant (i.e., specificity constants) were measured for
lipase PS immobilized inside the microreactor and for native lipase PS. The value inside the
microreactor was 800 times greater than that of the native lipase PS.
Introduction
As regards diffusion limitation in particular, if enzymes are
immobilized deep inside a matrix, the probability of the reactant
molecules encountering enzymes is limited because of the slow
molecular diffusion inside the support matrix. It is inferred
that enzymes immobilized near the matrix surface are mainly
exploited in the reactions. Therefore, if the diffusion limitation
in the support matrix is diminished, the apparent lipase activity
would be enhanced.
Enzymatic reactions have acquired increasing importance as
tools for fine chemical syntheses. Lipase exhibits an excellent
stereoselectivity and enantioselectivity in hydrolysis and acy-
lation reactions, and has been widely investigated over recent
1–3
decades. Lipase-catalyzed acylations are of great importance
for optical resolution in chemical syntheses; however, native
lipase is not uniformly dispersed in organic solvents because of
its poor solubility, which results in low enzymatic activity. It is
reported that the activity and enantioselectivity in the acylation
of chiral alcohols catalyzed by lipase PS can be altered in various
A microreactor with a small reaction space in the range from
15–17
100 mm to 1 mm is a useful tool for fine chemical synthesis.
One characteristic feature is that it has a large interfacial area
per unit volume. If a catalyst is coated on its wall, reactant
molecules can easily encounter the catalyst because of the short
diffusion distance inside the microreactor. Such a system can
be expected to be an efficient tool for heterogeneous catalytic
4,5
solvent systems. Many attempts have been made to improve the
4–9
lipase activity and enantioselectivity, and a common solution
is to immobilize it in support matrices, such as metal oxides, sol–
10–12
gel materials, and polymers.
Lipase is uniformly dispersed in
18
reactions. Microreactors have increasingly been employed for
a matrix by the immobilization, and can be used effectively in
organic solvent systems.
enzymatic reactions as high throughput analytical and synthetic
14,19
tools.
In these applications, enzymes were immobilized inside
Enzyme immobilization has commonly been employed for
microreactors with elegant techniques including biotin–avidin
13
large scale synthesis in both aqueous and organic solvents.
20–22
23,24
labeling techniques,
cross-linking enzyme aggregation,
Since immobilization simplifies the separation and reuse of
enzymes reducing chemical wastes, it is a valuable tool in
25–28
and use of metal oxide monolith/beads.
A simple and robust
enzyme immobilization method is desired for further use of
microreactors.
14
food, pharmaceutical, and chemical industries. However, by
comparison with native enzymes homogeneously dissolved in an
aqueous solution, the activity of enzymes in support matrices is
generally reduced. This is mainly attributed to the denaturation
of the enzymes and to the diffusion limitation of the reactants.
Recently, we have developed a microreactor that has a
29,30
mesoporous silica (MPS) thin film on its inner walls.
MPS,
which is typically synthesized with a silica precursor and
surfactant micelle templates, and possesses a highly ordered
31–34
pore structure.
Because of its uniform pores (about 7.5 nm
in diameter), MPS is suitable for use as a catalyst support,
National Institute of Advanced Industrial Science and Technology
35
(
AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
and has recently been used as an enzyme support matrix.
Several types of MPS powder exhibited high enzyme loading
E-mail: endo-akira@aist.go.jp; Fax: +81-29-861-4660;
Tel: +81-29-861-4653
36
37
38–43
capacities for horseradish peroxidase, catalase, and lipase.
It is also reported that this immobilization technique often
†
Electronic supplementary information (ESI) available: Estimation
of effective diffusivity inside mesoporous silica films. See DOI:
0.1039/b917374a
36,37
1
enhanced the thermal and chemical stabilities of enzymes.
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Therefore, the MPS film was provided inside the microreactor
as a support matrix. Importantly, the thin MPS film (less than
MPS thin films on QCM oscillators by dip coating with a
-
1
3.6 cm min withdrawing speed. The hydrophobic surfaces
were prepared with HMDS. The prepared MPS thin films were
exposed to HMDS vapor at room temperature for 2 h. After this
1
50 nm) could minimize the diffusion limitation within the
MPS support matrix, and enhance the apparent activity of
immobilized enzymes. In this context, a microreactor containing
MPS films is better adapted to enzymatic reactions in fine
chemical syntheses.
◦
exposure, the films were dried at 70 C to remove excess adsorbed
HMDS. In this process, the surface Si–OH groups of MPS were
45
converted into methyl terminals and became hydrophobic.
This study describes the immobilization of lipase PS in MPS
thin films inside a microreactor for use in the acylation of
Surface and cross-sectional images of the prepared MPS films
were acquired with a scanning electron microscope (SEM) at an
acceleration voltage of 0.8–1.5 kV (S-4800 FE-SEM: Hitachi
High-Technologies, Japan). For this observation, the micro-
capillary tubes were broken to reveal the MPS films coated on
their inner walls.
(
±)-1-phenylethanol with vinyl acetate. This paper has two main
parts. The first part describes the testing of several types of
MPS film appropriate for lipase immobilization, and the second
part evaluates the immobilized enzyme activity by comparison
with native lipase PS. The surface hydrophilicity and porous
structure of MPS films are discussed in the first part with a view
to achieving effective lipase immobilization. It is known that
In the lipase immobilization process, the developed microre-
-
1
actor was filled with a 10 mg mL lipase PS solution and kept
◦
at 5 C for 12 h to reach adsorption equilibrium. The lipase
the hydrophilicity of SiO
2
considerably alters the adsorption
solution was prepared by dissolving lipase PS SD in 20 mM
MES buffer solution controlled at pH 7. Prior to the experiment,
the microreactor was rinsed with the buffer solution for more
12,44
property of lipase.
The porous structure of MPS film affects
the access of reactant molecules to the immobilized lipase. These
two factors would play an important role in lipase activity. In the
second part, the activity of the immobilized lipase is quantified
by fitting to a Ping-Pong Bi Bi kinetic model for comparison
than 30 min to remove excess lipase. Subsequently, N gas was
2
fed through the microreactor to remove any remaining buffer
solution because the reactant solution is not soluble in water.
In the QCM analyses used to measure the amount of adsorbed
13
with native lipase.
-
1
lipase, 10 mg mL lipase solution was fed into the system where
it remained for 2 h. After flowing the buffer solution to remove
excess lipase near the surface, the mass change of the QCM
oscillator was monitored.
Experimental
All chemicals were used as received. For the MPS preparation,
ethanol, tetraethyl orthosilicate (TEOS), and hydrochloric acid
were obtained from Junsei Chemical Co. (Tokyo, Japan). F127
block copolymer supplied by BASF (Mount Olive, NJ, USA)
was used as a structure-directing agent. A borosilicate tube
Transesterification was carried out with lipase immobilized
inside the microreactor. All the experiments using the microre-
◦
actor were performed at 40 C in a continuous flow manner. The
reaction is expressed as follows:This model reaction catalyzed
(
0
Vitrocom, Mountain Lakes, NJ, USA) containing six bores
.20 mm in inner diameter and 30 cm long (3 mm outer diameter)
was used as a microreactor. Lipase PS SD and lipase PS IM
are products of Amano Pharmaceutical Co. Note that lipase
PS IM is a commercial formulation of lipase PS immobilized
on SiO (diatomaceous earth). For a buffer solution, 2-(N-
2
morpholino)ethanesulfonic acid (MES) was obtained from
Dojin Chemical (Kumamoto, Japan). For the transesterifi-
cation, (±)-1-phenylethanol (PE) and (S)-(-)-1-phenylethanol
by lipase PS is known as a route to enantiomerically pure
compounds under mild conditions. Because of its high activity
3
and selectivity, the reaction is frequently discussed in the field
4,46
(
(S)-PE) were purchased from Wako Pure Chemical Industries
of green chemistry. A mixture of (±)-PE and VA dissolved in
-
1
Ltd. (Osaka, Japan); (±)-1-phenylethylacetate (PEA) was pur-
iPr O was fed at a rate ranging from 0.3 to 24 mL min by using a
2
chased from Merck Schuchardt OHG (Hohenbrunn, Germany);
syringe pump (PHD2000, Harvard Apparatus, Holliston, MA,
USA). The initial concentration was ranging from 5 to 26 mM
for PE and from 5 to 50 mM for VA, respectively. The effluent
was collected to measure its concentration with an HPLC
(Shimadzu LC–10A, Kyoto, Japan) equipped with a Chiralcel
OD column (Daicel Chemical Industries, Tokyo, Japan). The
UV detector was set at 254 nm, and the column temperature was
vinyl acetate (VA) and diisopropyl ether (iPr O) were ob-
2
tained from Kishida Chemical Co. (Osaka, Japan); 1,1,1,3,3,3-
hexamethyldisilazane (HMDS) was purchased from Aldrich
(
Milwaukee, WI, USA).
The preparation of MPS thin films inside microreactors
29,30
has been detailed elsewhere.
Two different MPS struc-
◦
tures were formed in the present study. The molar ratios of
TEOS, H O, HCl, EtOH, and F127 in precursor solutions
were 1 : 9.2 : 0.042 : 51 : 0.0041 for 3D cubic MPS film and
: 9.2 : 0.021 : 40 : 0.0072 for 2D hexagonal film. The inner walls
held at 30 C. The mobile phase was hexane/2-propanol (6/1),
-
1
2
and the flow rate was 1 mL min . Reference compounds in-
cluding (±)-1-phenylethanol, (S)-(-)-1-phenylethanol, and (±)-
1-phenylethylacetate were used to identify the retention time of
the products.
1
of micro-capillary tubes were coated with these precursor
solutions. In fact, it is difficult to measure the amount of
adsorption inside microreactors directly. Accordingly, a quartz
microbalance system (QCM: Q-Sense D300, G o¨ teborg, Sweden)
was used for measuring the amount of lipase adsorbed on the
MPS films. The same precursor solutions were used to form
Additional batch experiments were conducted using lipase PS
SD and lipase PS IM to evaluate enzyme activity. The mixture
of PE and VA dissolved in iPr O (3 mL) was added to a 13.5 mL
2
glass vial containing lipase PS SD (30 mg) or lipase PS IM (5 mg),
◦
and was then stirred (600 rpm) at 40 C. The initial concentration
3
32 | Green Chem., 2010, 12, 331–337
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of PE tested in these batch experiments was ranging from 6 to
thickness is about 125 nm and that the pore channel size is about
2
5
6 mM; the initial concentration of VA was ranging from 5.7 to
7 mM. After completion of the reaction time, the suspension
7.5 nm, which is almost the same as the pore size of 3D cubic
film. We confirmed that ordered pore structures were present
inside the microreactor. We also confirmed that the films on the
QCM oscillators had the same structures as those inside the
microreactor.
was quickly filtered through a cellulose acetate filter (0.5 mm,
Advantec DISMIC-13, Tokyo, Japan) and then was injected
into the HPLC system.
The amount of lipase adsorbed on the MPS films on QCM
oscillators was measured in order to understand the properties
of support matrices suitable for lipase immobilization. We
investigated MPS films with and without HMDS hydrophobic
treatment. Hereafter, we refer to HMDS-treated 3D cubic
and 2D hexagonal films as H-cubic and H-hexagonal films,
respectively. The mass change (DM) of QCM oscillators coated
with MPS films was monitored after they were exposed to
lipase solution (Fig. 2). Fig. 2 shows that lipase adsorption
increased rapidly and reached equilibrium after exposure to
lipase solution. Importantly, DM remained constant after it
was rinsed with buffer solution, which indicates that little lipase
leached from the MPS films. Although several reports mention
that a strong interaction between enzyme and silica is required in
Results and discussion
MPS films for lipase PS immobilization
The first part of this study focuses on the influence of the
porous structure and hydrophilicity of MPS thin films on the
amount and activity of adsorbed lipase. Fig. 1 shows SEM
images of MPS thin films that were formed on the inner wall
of the microreactor. Two forms of MPS were prepared for
the investigation: 3D-cubic (Fig. 1a and b) and 2D-hexagonal
(
Fig. 1c and d). A major difference between two structures was
the existence of open mesopores on the surface. Uniform open
pores about 7.5 nm in diameter are aligned in an orderly way
with 3-fold symmetry on the surface (Fig. 1a). This is a typical
the immobilization process to minimize the amount of enzyme
30,34
feature of a 3D cubic structure.
As shown in Fig. 1b, the
40,47
leaching out from silica,
the MPS films exhibited excellent
film thickness is about 45 nm. With the 2D hexagonal film
shown in Fig. 1c, the pore channels are aligned parallel to the
surface forming a swirling pattern with few open pores (dark
spots in the figure) on the surface. Fig. 1d shows that the film
affinity to lipase PS. The final values of DM are also listed
in Table 1. A comparison of the final DM values for MPS
films showed that lipase adsorbed more on HMDS-treated films
than on untreated films. The lipase adsorption on H-cubic film
-
2
(
(
(
1.55 mg cm ) was about 9 times that on the untreated film
-
2
0.173 mg cm ), while the adsorption on H-hexagonal film
-
2
2.12 mg cm ) was about 20 times that on the untreated film
Fig. 2 Amount of lipase adsorbed in MPS films on QCM oscillators:
(from top to bottom) solid line: H-hexagonal, long-dashed line: H-hubic,
short-dashed line: cubic, dotted line: hexagonal. At 2 min, lipase solution
was supplied to the QCM system. Every 10 min after 122 min, the buffer
solution was supplied to remove excess lipase.
Fig. 1 Structures of mesoporous thin films on the inner wall of
microreactors: (a) Surface image of 3D cubic; (b) cross-sectional image
of 3D cubic; (c) surface image of 2D hexagonal, (d) cross section of 2D
hexagonal. Scale bar: 100 nm.
Table 1 Amount and activity of lipase adsorbed on 4 types of MPS thin film
-2a
b
b
b
c
c
d
Type of MPS
DM/mg cm
(R)-PEA/mM
(R)-PE/mM
(S)-PE/mM
i-(R)-PE/mM
i-(S)-PE/mM
Yield [%]
Cubic
Hexagonal
H-Cubic
H-Hexagonal
H-Cubic
0.173
0.102
1.55
2.12
—
0.272
0.202
9.53
6.64
11.2
12.9
13.1
5.44
8.31
1.17
12.9
12.4
14.9
14.8
12.5
13.0
13.0
14.9
14.9
12.1
12.9
12.9
14.9
14.9
12.1
2.10
1.56
63.9
44.7
92.5
e
a
b
c
Mass change (DM) was calculated from frequency shift (Df ) of QCM based on Sauerbrey equation. The concentration in the effluent. The
d
e
concentration in the influent. Ratio of the concentration of (R)-PEA in the effluent and the initial concentration of (R)-PE. Microreactor
-1
-1
experiment was at a flow rate of 0.3 mL min . Other experiments were at 1.2 mL min . When the flow rate is small in this system, the reaction proceeds
further because of long contacting time.
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-
2
(
0.102 mg cm ). It is clear that lipase was preferentially adsorbed
hexagonal film. The H-hexagonal film has a limited number
of open pores on the surface and appears to provide poor
accessibility to its pores through its surface compared with H-
on the hydrophobic films. This agrees with other reports on
silica and MPS.
electrostatics, hydrogen bonding, hydrophobic interactions, and
12,39,44
While several types of interaction including
30,34
cubic film (see Fig. 1c).
In some cases, there are micropores
48
van der Waals interactions govern protein adsorptions, the
hydrophobic interaction dominates in the lipase adsorption in
MPS. Furthermore, the QCM results showed that more lipase
was adsorbed on H-hexagonal film than on H-cubic film. It is
simply because the thickness of H-hexagonal film is greater than
that of H-cubic film. As shown in Fig. 1b and d, the hexagonal
film (125 nm) was thicker than the cubic film (45 nm). Because
thicker films should have a larger pore volume, the H-hexagonal
film had a greater lipase adsorption capacity than the H-cubic
film. From the DM values and the size of lipase, we calculated the
total volume of adsorbed lipase and compared it with the size of
the pore volume. From this estimation, lipase was adsorbed on
almost the entire H-cubic film from top to bottom, while an open
space still remained in the H-hexagonal film. Therefore, lipase
was adsorbed more on the H-hexagonal film because it even
migrated inside the narrow pore channels of the H-hexagonal
film during the long immobilization process.
(smaller than 2 nm) on the walls of mesopore channels. However,
the diffusion of reactant molecules through such micropores is
presumably small compared with the diffusion through the open
mesopores in Fig. 1a. Unlike the H-hexagonal film, the H-cubic
film has many open mesopores on its surface, and the reactant
molecules have good accessibility to the lipase immobilized
inside the mesopores. Since the reaction was conducted in a
continuous flow manner, its reaction rate was greatly affected
by diffusion limitation. Hence, the reaction rate of immobilized
lipase is correlated with the MPS film structure in terms of
reactant molecules’ accessibility to lipase inside the pores. Of
the MPS films tested in this study, the H-cubic film is the most
suitable for transesterification with immobilized lipase.
In our preliminary experiments, lipase PS SD and lipase PS
IM showed good enantioselectivity in batch reactions. However,
it should be noted that the enantioselectivity of lipase PS
is dependent on its solvent environment and is sometimes
1,4
The activity of the adsorbed lipase was also compared
with four types of MPS film coated inside the microreactors.
Transesterification was carried out in a continuous flow manner
altered by the addition of ionic liquids. In this context,
the enantioselectivity in the experiments was judged from the
(S)-PEA and (S)-PE concentrations in Table 1. In fact, the
concentration of (S)-PEA was lower than the detection limit;
the change in the (S)-PE concentration was negligible. Hence, in
all cases, the enantiomeric excess values were higher than 99%.
For the H-cubic film, the enantioselectivity was high even at a
yield of 92.5% in an additional experiment with a flow rate of
-
1 49
at a rate of 1.2 mL min . The measured concentration
of the effluent flow is summarized in Table 1. The product
concentrations, (R)-PEA, for the HMDS-treated films (9.53 mM
for H-cubic and 6.64 mM for H-hexagonal) were about 35 times
greater than that for the bare films (0.272 mM for cubic and
-
1
0
.202 mM for hexagonal). This supports the QCM results
0.3 mL min . Thus, the lipase immobilized in the MPS films
showing that more lipase was adsorbed on the hydrophobic
surface than on the hydrophilic surface. The result also suggests
that lipase immobilized on the hydrophobic surface maintained
good enzymatic activity and that denaturationvia the adsorption
process was not significant in the current system. Furthermore,
it should be noted that while H-cubic film had only 9 times more
lipase adsorption than the untreated cubic film, it exhibited a
maintains good enantioselectivity.
Evaluation of immobilized lipase PS
The H-cubic film exhibited a good capacity for lipase immobi-
lization based on an activity test for four types of MPS film.
The second part of this study focuses on the characteristics of
lipase immobilized in the H-cubic film. The reactant solution
was continuously fed into a microreactor containing lipase PS at
3
5-fold increment in the product concentration. In other words,
lipase adsorbed on H-cubic film was more active than that on
the untreated cubic film. It is reported that the lipase active site
is covered by a lid domain and that the lid moves away when
-
1
a rate of 3 mL min for 36 h, and the product concentration in the
effluent solution was monitored to determine its stability (Fig. 3).
The concentration after the first hour was 6.21 mM, which
corresponds to a yield of 51% in a contact time of 18.8 min. The
50
lipase is exposed to hydrophobic environments. An open form
of lipase is thought to exhibit good activity. The hydrophobic
treatment in the present study also had a similar effect on the
transesterification catalyzed by lipase. Indeed, both H-cubic
and H-hexagonal films had similar acceleration effects on the
reaction. The results indicate that the hydrophobic treatment of
MPS thin film enhanced the catalytic activity of immobilized
lipase as well as the amount of adsorption.
We discuss the effect of MPS structure on the activity of
immobilized lipase by comparing H-cubic and H-hexagonal
films. As shown in Table 1, the (R)-PEA concentration of the
H-hexagonal film (6.64 mM) was 30% less than that of the
H-cubic film (9.53 mM), whereas the lipase adsorption of the
-
2
H-hexagonal film (2.12 mg cm ) was 70% more than that of
-
2
the H-cubic film (1.55 mg cm ). Interestingly, the reaction rate
of lipase immobilized in the H-hexagonal film was slower than
that in the H-cubic film. We speculate that the slow reaction rate
was due to reactant molecules’ access to the lipase inside H-
Fig. 3 Changes in the product concentration in the effluent solution
with time in long-term continuous test. The reactant solution (C(R)-PE
12.1 mM, C
=
(
S)-PE
= 12.1 mM, C = 50 mM) was continuously fed at
VA
-1
◦
3 mL min over 36 h. The temperature of the reactor was kept at 40 C.
3
34 | Green Chem., 2010, 12, 331–337
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concentration at 8 h was 5.71 mM and the final concentration at
51
3
6 h was 4.94 mM, which was about 80% of the original value.
The results indicate that the reaction rate slowly decreased with
time. Since a small amount of lipase was leached out in the QCM
experiments, the decrease in the reaction rate was largely due to
the natural decay of lipase activity. It is well known that lipase
41,52
loses its activity in organic solvents.
Amano Pharmaceutical
Co. also reported that lipase PS IM loses about 20% of its activity
◦
after being stirred in iPr
2
O at 25 C for 24 h. Therefore, the lipase
immobilized in the H-cubic film provided fairly good stability,
based on the result showing that it maintained about 80% of its
◦
original activity after 36 h of continuous reaction at 40 C.
The enzymatic activity of lipase immobilized in MPS films
was quantified for evaluation through a comparison with native
lipase and a commercial immobilized lipase PS. Since the
MPS film coated inside the microreactor was designed for
continuous operation, we conducted tests with various reactant
-
1
concentrations at a flow rate of 24 mL min , and calculated
the reaction rates from the product concentrations. Compared
with the flow rate in the previously described activity test
-
1
-1
(
1.2 mL min ), the rate in this experiment is fast (24 mL min ).
Under this condition, the change in the reactant concentration
is very small because of the short contact time. This allows us
to assume that the reactant concentration is constant across
the microreactor and then to calculate the reaction rates directly
29
from product concentrations and flow rates. It should be noted
that the rates obtained with this method are equivalent to the
initial rates in the batch experiments.
Fig. 4a shows the reaction rates as a function of VA
concentration at (R)-PE concentrations, C(R)-PE of 2.5, 5, 6,
and 13 mM. The reaction rates gradually increase with VA
concentration and reach a plateau at high VA concentrations.
This feature is observed for all PE concentrations. For further
analysis, the results were re-plotted in Lineweaver–Burk form,
where the reciprocals of the reaction rates were plotted against
the reciprocal of the VA concentration, CVA (Fig. 4b). Data sets at
a constant C(R)-PE form straight parallel lines. We also confirmed
that the y-intercepts of the straight lines are proportional to
the reciprocal of C(R)-PE. These are typical features representing
Fig. 4 (a) Transesterification experiments as a function of VA concen-
tration. The solid lines are the fitted results based on the Ping-Pong Bi Bi
mechanism. In the graph, C(R)-PE is the concentration of (R)-PE in mM.
ꢀ
: C(R)-PE = 13 mM, D: C(R)-PE = 6 mM, ᭹: C(R)-PE = 5 mM,
ꢀ
: C(R)-PE
=
2.5 mM. (b) Lineweaver–Burk plot of the transesterification experiments
in Fig. 4a. Dotted lines are guides to the eye.
substrate concentrations (CVA and C(R)-PE) were set at very low
54,55
values, eqn (1) can be simplified as follows:
(
2)
13
the Ping-Pong Bi Bi mechanism. In this transesterification,
VA first binds to lipase to give an intermediate form that then
reacts with PE to yield the product, PEA. It is known that PE
sometimes acts as a competitive inhibitor in the first step of this
In eqn (2), it is estimated that 1/Vmax is far smaller than
VA /VmaxCVA and KPE/VmaxC(R)-PE. The specificity constants,
K
53–55
reaction.
The current results show that the rate increased
which correspond to Vmax/KVA and Vmax/KPE, indicate the
with the PE concentration (Fig. 4a), and suggest that PE
inhibition was minimal under these conditions. This is because
we set the initial reactant concentrations at relatively low values
in order to quantify the initial activity of lipase immobilized
in MPS films in the absence of competitive inhibition. While
many kinetic forms have been proposed for the interpretation
efficiency of an enzyme that converts a substrate into a product.
Hence, instead of estimating the individual parameters of Vmax
,
K
PE, and KVA , the specificity constants were obtained by fitting
the results to eqn (2) to evaluate the immobilized lipase activity.
In addition, the slope of the straight lines in the Lineweaver–
Burk plot corresponds to KVA /Vmax, which is the reciprocal
of the specificity constant (Fig. 4b). To estimate commercial
lipase activity, the initial reaction rates were measured in batch
experiments using lipase PS SD and lipase PS IM. The results
of the batch experiments were also fitted to eqn (2) in the
same manner. The obtained specificity constants of immobilized
lipase, lipase PS SD, and lipase PS IM are summarized in Table 2.
The values of Vmax/KVA and Vmax/KPE for lipase immobilized
in the MPS film are roughly the same, which indicates that
the Ping-Pong constants, KVA and KPE are not very different
56–58
of transesterification,
the Ping-Pong Bi Bi mechanism in
the absence of competitive inhibition was suitable for acquiring
kinetic parameters in this study and is expressed as follow:
(
1)
where Vmax is the maximum velocity (mol s-
1
g
lipase-1); KVA and
K
PE are the Ping-Pong constants for VA and PE (M). Since the
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Table 2 Specificity constants of lipase PS
applicability to enantioselective chemical reactions catalyzed by
immobilized enzymes.
Type of lipase
V
max/KVA [L s-
1
g
lipase-1]
V
max/KPE [L s-1 lipase-1]
g
-
3
6
4
-3
Immobilized
Lipase PS SD
Lipase PS IM
4.50 ¥ 10-
5.71 ¥ 10
3.86 ¥ 10
6.85 ¥ 10
3.08 ¥ 10
Acknowledgements
-6
-4
-
1.17 ¥ 10
This study was funded by “Development of Microspace and
Nanospace Reaction Environment Technology for Functional
Materials” project (NEDO, Japan). We thank Dr Toshiyuki
Kanamori for use of the QCM system and Dr Toshiyuki Takagi,
Dr Yasumasa Takenaka and Dr Katsuyuki Iwanami at AIST
from each other. The specificity constant Vmax/KVA for lipase
immobilized in the MPS film is greater than that for native
lipase PS SD by a factor of nearly 800. As described in the
introduction, lipase PS was not dissolved in organic solvents
and was not equally dispersed in the solution. This low solubility
caused the native lipase activity to be low in organic solutions.
Hence, the results suggest that the lipase immobilized in the
MPS film encountered reactant molecules with an increased
frequency, which led to excellent enzymatic activity in the trans-
esterification. The specificity constant of lipase PS IM in Table 2
was estimated for the total weight which includes diatomaceous
earth and lipase because the immobilization percentage was not
provided. Although the specificity constant of lipase PS IM is
not directly comparable, the specificity constant for the lipase
(
Tsukuba, Japan) for fruitful discussion.
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