Paper-immobilized enzyme as a green microstructured catalyst

Article abstract of DOI:10.1039/c2jm30759f

The facile and direct introduction of methacryloxy groups into cellulose paper was carried out using a silane coupling technique, leading to the improvement of hydrophobicity and both dry and wet physical strengths of the paper. Immobilization of lipase enzymes onto the methacrylate-modified paper was then accomplished, possibly due to hydrophobic interaction. The as-prepared immobilized lipase on methacrylate-modified paper possessed paper-specific practical utility. During a batch process for the nonaqueous transesterification between 1-phenylethanol and vinyl acetate to produce 1-phenylethylacetate, the paper-immobilized lipase showed high catalytic activity, selectivity and reusability, suggesting that the methacryloxy groups introduced into the cellulose paper played a key role in the hyperactivation of lipases. In addition, a higher productivity of 1-phenylethylacetate was achieved in a continuous flow reaction system than in the batch system, indicating that the interconnected porous microstructure of the paper provided favorable flow paths for the reactant solution. Thus, the paper-immobilized enzyme is expected to offer a green catalytic material for the effective production of useful chemicals.

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PAPER
Paper-immobilized enzyme as a green microstructured catalyst†
a
bc
a
Hirotaka Koga,* Takuya Kitaoka and Akira Isogai
Received 8th February 2012, Accepted 3rd April 2012
DOI: 10.1039/c2jm30759f
The facile and direct introduction of methacryloxy groups into cellulose paper was carried out using
a silane coupling technique, leading to the improvement of hydrophobicity and both dry and wet
physical strengths of the paper. Immobilization of lipase enzymes onto the methacrylate-modified
paper was then accomplished, possibly due to hydrophobic interaction. The as-prepared immobilized
lipase on methacrylate-modified paper possessed paper-specific practical utility. During a batch process
for the nonaqueous transesterification between 1-phenylethanol and vinyl acetate to produce 1-
phenylethylacetate, the paper-immobilized lipase showed high catalytic activity, selectivity and
reusability, suggesting that the methacryloxy groups introduced into the cellulose paper played a key
role in the hyperactivation of lipases. In addition, a higher productivity of 1-phenylethylacetate was
achieved in a continuous flow reaction system than in the batch system, indicating that the
interconnected porous microstructure of the paper provided favorable flow paths for the reactant
solution. Thus, the paper-immobilized enzyme is expected to offer a green catalytic material for the
effective production of useful chemicals.
Introduction
Immobilized enzymes have been prepared through various
interactions, such as physical adsorption, hydrophobic interac-
16
tion, covalent and electrovalent bonding. Because lipases have
Enzymes have received increasing interest as green catalysts for
a wide range of chemical industries because of their high catalytic
relatively high hydrophobicity, simple adsorption of lipases on
1,2
activity and selectivity under mild conditions. Amongst the
many enzymes in existence, lipases (glycerol ester hydrolases
E.C.3.1.1.3) are known to catalyze the hydrolysis of fats in their
natural environment. In recent years, lipase-catalyzed reactions
in nonaqueous media, such as transesterification, esterification,
suitably hydrophobic supports is regarded as one of the most
15,17–20
effective approaches.
The hydrophobic surface of supports
17,18
leads to hyperactivation of lipases via interfacial adsorption,
because the change in conformation of lipases caused by
hydrophobic interaction provides efficient accessibility of
substrates to their active centers. For this reason, the surface
modification of supporting materials has been conducted to
obtain suitable hydrophobicity, and ceramic supports modified
3
aminolysis, acyl exchange and thiotransesterification, have been
extensively studied for synthesis of many useful chemicals for
4–6
food, cosmetic, pharmaceutical and biodiesel applications.
However, enzymes, including lipases, are frequently unstable in
7
nonaqueous media and easily aggregate and deactivate. To solve
with methacryloxy groups have been reported as effective sup-
10,11
porting materials for lipases.
In addition, the structural
this problem, enzymes are often immobilized on various sup-
10,11
design of porous supports has been intensely studied to enable
8
,9
porting materials, such as silica, ceramics,
13,14
carbonaceous
their use as microstructured flow reactors to efficiently and
11,21–29
1
2
materials, polymers and resins.
Such immobilized enzymes
15,16
provide reusability as well as stability in nonaqueous media
continuously obtain target products.
However, there is still
a
requirement to develop high-performance immobilized
16
and offer advantages in the isolation of products.
enzymes with excellent practical utility.
Cellulose (b-1,4-D-glucopyranose polymer), the principal
ingredient in plant-based biomass, is the most common and
a
30
abundant organic polymer. From the viewpoint of sustainable
Department of Biomaterials Sciences, Graduate School of Agricultural
and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan. E-mail: ahkoga@mail.ecc.u-tokyo.ac.jp; Fax:
development, the active use of cellulosic resources is of growing
importance, and many researchers have energetically developed
+
b
81-3-5841-5271; Tel: +81-3-5841-5271
31–41
new cellulose-based materials with various functions.
Paper
Department of Agro-environmental Sciences, Graduate School of
Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1
Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
is one of the most common cellulosic products, traditionally used
in our daily life for various purposes such as writing, printing and
packaging. Paper materials are low cost, lightweight, flexible
and easy to handle, and thus have excellent practical utility.
In addition, several of our previous studies indicate that the
c
Biotron Application Center, Kyushu University, 6-10-1 Hakozaki,
Higashi-ku, Fukuoka 812-8581, Japan
†
1
Electronic supplementary information (ESI) available. See DOI:
0.1039/c2jm30759f
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paper-specific interconnected porous microstructure derived
from fiber networks can provide a favorable reaction environ-
ment, especially in flow-type catalytic reactions, allowing for the
reduced pressure. The obtained paper was thermally treated at
ꢁ
110 C for 3 h, then thoroughly washed with ethanol and dried at
room temperature.
42–45
effective diffusion of heat and reactants.
Therefore, paper is
expected to serve both as a versatile support material and as an
effective microstructured flow reactor for various catalysts,
including enzymes.
Immobilization of lipase on methacrylate-modified paper
Immobilization of lipase on original (unmodified) or methacry-
late-modified cellulose paper was performed as follows. Crude
lipase powders (10 mg) were suspended in phosphate buffer
solution (pH ¼ 6.86, ionic strength: 0.1 M, 10 ml) and a piece of
original or methacrylate-modified cellulose paper was then
immersed in the lipase solution with insoluble components,
followed by stirring (150 rpm) by using an orbital platform
shaker (Rotamax 120, Heidolph Instruments GmbH & Co. KG)
We have recently reported the direct introduction of amino
groups into cellulose paper using a silane coupling technique
through the condensation reaction between the Si–OH of the
organofunctional silane coupling agent and the C–OH of cellu-
lose, which we refer to as the in situ modification of cellulose
37
paper with amino groups. The as-prepared paper material,
denoted amine-modified paper, had excellent handling conve-
nience and demonstrated high catalytic efficiency and reusability
in the Knoevenagel condensation reaction. The silane coupling
technique has great potential for introducing an array of func-
tional groups into cellulose paper and to develop newly func-
tionalized paper materials. The chemically modified cellulose
paper is expected to offer a promising supporting material for
enzymes.
ꢁ
at 23 C for 12 h. The treated paper was then removed from the
solution, thoroughly washed with phosphate buffer solution
(pH ¼ 6.86, ionic strength: 0.1 M, 10 ml) at a stirring rate of 150
rpm for 3 h to remove unwanted insoluble components adsorbed
onto the paper, and dried in a desiccator at room temperature.
The lipase content in the as-prepared paper was measured in part
25,47
according to the method described by previous reports:
In this study, the in situ modification of cellulose paper with
methacryloxy groups as binding sites for lipases was investigated.
The physical properties, i.e. mechanical strength and hydro-
phobicity, of the as-prepared methacrylate-modified paper were
evaluated. The immobilization of lipase on methacrylate-modi-
fied paper was conducted and the catalytic performance of the
immobilized lipase on methacrylate-modified paper was then
investigated for a nonaqueous transesterification reaction in both
batch and flow modes.
enzymatic activity of the lipase solutions (supernatant, not
paper) before and after soaking treatment of the paper was
determined using a colorimetric assay based on the hydrolysis of
4-nitrophenyl acetate, and the amount of lipase units adsorbed
onto the papers was estimated by subtraction. In summary, the
lipase solution after the removal of the paper was mixed with
the buffer solution that was used for washing the paper. Then the
mixture was diluted 11.4-fold with fresh buffer solution. Subse-
quently, the lipase solution (2.85 ml) was added to an acetonitrile
solution of 4-nitrophenyl acetate (1.0 mM, 0.15 ml). The reaction
progress of the hydrolysis of 4-nitrophenyl acetate to produce
Experimental
4
-nitrophenol was monitored at room temperature by UV-Vis
Materials
absorption spectroscopy (V-670 spectrophotometer, JASCO);
the enzymatic activity of the lipase solution was determined by
measuring the change in absorbance at 400 nm, which was
derived from 4-nitrophenol, as a function of time. The original
lipase solution before soaking treatment of the paper samples
was also subjected to the colorimetric assay, and the amount of
lipase units adsorbed onto the papers was estimated by
subtraction.
Cellulose filter paper (ADVANTEC No. 5A, cotton linter
cellulose content >99 wt%) was purchased from Toyo Roshi
Kaisha, Ltd., Japan, and was cut into circular pieces (ca. 90 mg,
3
3 mm in diameter) before use. 3-(Trimethoxysilyl)propyl
methacrylate (>98.0%, Tokyo Chemical Industry Co., Ltd.,
Japan, see ESI, Fig. S1†) was used as a silane coupling agent.
Lipase from Burkholderia cepacia (Amano Lipase PS, lipase
ꢀ1
protein content ca. 5.6 mg mg (ref. 13 and 46)) was obtained
from Sigma-Aldrich Co. Ltd. (St Louis, MO, USA). (R,S)-1-
Phenylethanol (>98.0%), (R)-1-phenylethanol (>98.0%), (S)-1-
phenylethanol (>98.0%), vinyl acetate monomer (>99.0%), iso-
propyl ether (>99.0%) and 4-nitrophenyl acetate (>98.0%) were
obtained from Tokyo Chemical Industry Co., Ltd., Japan. All
reagents were used without further purification.
Nonaqueous transesterification reaction
The nonaqueous transesterification reaction was carried out in
both batch and flow modes. In batch mode, (R,S)-1-phenyl-
ethanol (0.41 mmol) and vinyl acetate (0.62 mmol) were dis-
solved in isopropyl ether (10 ml). A piece of immobilized lipase
on original or methacrylate-modified paper (33 mm in diameter)
was then immersed in the solution. The reaction was carried out
in a closed glass vial (SV-50A, NICHIDEN-RIKA GLASS Co.
Preparation of methacrylate-modified paper by the silane
coupling technique
ꢁ
Ltd., 40 mm in diameter, 75 mm in height) at 23 C with and
3
-(Trimethoxysilyl)propyl methacrylate (100 mg) was added to
without continuous stirring by an orbital platform shaker. At
a given time, the reaction solution was analyzed to determine the
concentration of the desired product, 1-phenylethylacetate, using
a gas chromatograph with a (GC)-flame ionization detector,
equipped with a TC-1 column (0.25 mm ꢂ 30 m, GL Sciences
Inc.). As a control, free lipase powders were also subjected to the
performance test. One unit of lipase activity (U) was defined as
an acetic acid aqueous solution (pH ¼ ca. 4, 10 ml), and the
mixture was vigorously stirred for 30 min. Then it was hydro-
lyzed to form reactive silanol groups and thus was dissolved in
the solution. Subsequently, a piece of cellulose paper was
immersed in the resulting solution for 2 h, followed by evapo-
ꢁ
ration of an acetic acid aqueous solution at 40 C for 3 h under
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ꢁ
the amount of enzyme required to produce 1 mmol of 1-phenyl-
ethylacetate per minute, which was determined on the basis of the
amount produced over 1 h. To evaluate enantioselectivity, (R)-1-
phenylethanol or (S)-1-phenylethanol was used as a substrate
instead of (R,S)-1-phenylethanol. Reusability of immobilized
lipase on methacrylate-modified paper was evaluated by a three-
cycle test; the sequential procedures of the transesterification
reaction (5 h), thorough washing with isopropyl ether and drying
at room temperature were conducted in each cycle of the test. In
flow mode, immobilized lipase on methacrylate-modified paper
was cut into fourteen circular discs (9.0 mm in diameter) and the
lipase content set equal to that used in the batch mode tests.
The papers were vertically stacked (ca. 3.1 mm in thickness)
and tightly packed into a syringe (Terumo syringe SS-02LZ,
TERUMO, ca. 9.1 mm in diameter) equipped with a silicon tube.
Subsequently, the substrate solution was fed into the paper layer
acid aqueous solution at 40 C for 3 h under reduced pressure,
thermal treatment and washing with ethanol. Fig. 1 profiles the
FT-IR spectra of the original cellulose paper and the cellulose
paper treated with 3-(trimethoxysilyl)propyl methacrylate. When
the cellulose paper was treated with 3-(trimethoxysilyl)propyl
methacrylate, the characteristic bands derived from C]C and
ꢀ1
ꢀ1
C]O stretching appeared at ca. 1640 cm and ca. 1720 cm ,
respectively (Fig. 1b). In addition, CH and CH stretching
vibration bands were observed at ca. 2950 cm and ca.
3
2
ꢀ1
ꢀ1
48
2930 cm , respectively. These results indicate the presence of
methacryloxy groups. As shown in Fig. 2, XRF spectra
confirmed introduction of Si to the paper after treatment. Thus,
it was suggested that cellulose paper was successfully modified
with methacryloxy groups by the silane coupling technique.
Previous reports concerning the silane coupling treatment of
37,49
cellulose
suggest that the successful direct introduction of
ꢀ1
at a constant flow rate of 0.1–2.0 ml min using a syringe pump
Econoflo, Harvard Apparatus). Productivity was calculated as
methacryloxy groups into cellulose paper was due to the
condensation reaction between Si–OH of silane coupling agents
and C–OH of cellulose. Fig. 3 shows SEM images of original and
methacrylate-modified paper. The paper-specific interconnected
porous microstructure remained almost unchanged even after
the silane coupling treatment. However, the surface morphology
of the cellulose fibers changed after the modification of meth-
acryloxy groups, suggesting that silane coupling agents were
coated on their surfaces. In addition, XRD analyses confirmed
that the crystal structure of cellulose I (ref. 50) was maintained
after silane coupling (see ESI, Fig. S2†). Thus, these results
suggest that methacryloxy groups were introduced into the
crystal surfaces of cellulose fibers.
(
the amount of product (1-phenylethylacetate) per minute per mg
lipase protein.
Analyses
Fourier transform infrared attenuated total reflection (FT-IR/
ATR) spectra were obtained using a FT/IR-6100 instrument
(
JASCO). A diamond prism was used as an internal reflecting
element for FT-IR/ATR analysis. X-ray fluorescence (XRF)
analysis was carried out using MESA-500 apparatus
HORIBA). Surface observation of as-prepared papers was
a
(
conducted using a scanning electron microscope (SEM, S-4000,
Hitachi, Ltd.) after osmium sputtering. X-ray diffraction (XRD)
patterns were recorded using a Rigaku RINT 2000 with Ni-
Table 1 displays the physical properties of unmodified and
methacrylate-modified cellulose paper. The weight of cellulose
paper increased by ca. 27 mg after the silane coupling treatment,
indicating that the content of silane coupling agents in the paper
was about 24 wt%. Then the methacrylate content in the paper
ꢀ
filtered CuKa radiation (l ¼ 1.5418 A) and scanning angle (2q) in
ꢁ
the range 10–30 , at 40 kV voltage and 40 mA current. For tensile
strength measurements, paper samples were cut into rectangular
pieces (5 mm in width, 20 mm in length). Tensile strengths and
ꢀ1
was roughly estimated at ca. 1.15 mmol g by the change in
paper weight. Methacrylate-modified paper was lightweight (ca.
ꢀ3
ꢁ
Young’s moduli of the papers were evaluated at 23 C and 50%
0
.6 g cm ). In addition, a ca. 20 mm increase in paper thickness
relative humidity using a Shimadzu EZ-TEST instrument
was observed, possibly caused by coating of silane coupling
agents on the cellulose fiber surfaces. The dry strength of cellu-
lose paper is derived from intra- and inter-fiber hydrogen bonds
ꢀ1
equipped with a 500 N load cell (pulling rate: 1.0 mm min , span
length: 10 mm). Wet strengths were measured after soaking in
deionized water for 2.5 h. The surface hydrophobicity of the
papers was evaluated by measuring the contact angle of water
with a DropMaster 500 contact angle meter (Kyowa Interface
Science Co. Ltd.) using the sessile drop technique.
Results and discussion
Preparation and characterization of methacrylate-modified
paper
In situ modification of cellulose paper with methacryloxy groups
was carried out using a silane coupling technique. 3-(Trime-
thoxysilyl)propyl methacrylate used as a silane coupling agent in
this study was insoluble in pure water. Therefore, it was added to
an acetic acid aqueous solution (pH ¼ ca. 4), and the mixture was
vigorously stirred for 30 min. Then, methoxy groups of 3-(tri-
methoxysilyl)propyl methacrylate were hydrolyzed to form sila-
nol groups (Si–OH) and to afford the aqueous solution.
Subsequently, a piece of cellulose paper was immersed in the
resulting solution for 2 h, followed by evaporation of an acetic
Fig. 1 FT-IR spectra of (a) original cellulose paper and (b) cellulose
paper treated with 3-(trimethoxysilyl)propyl methacrylate.
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between Si–OH groups, leading to the improvement of the
physical strength of the paper. Fig. 4 shows optical images of
water droplets on original and methacrylate-modified paper. The
contact angle of water droplets on cellulose paper increased from
ꢁ
ꢁ
0
to ca. 110 with silane coupling, indicating a dramatic
improvement in hydrophobicity. This result can be ascribed to
hydrophobic moieties, the propyl and methacryloxy groups of
silane coupling agents (see ESI, Fig. S1†), introduced into
hydrophilic cellulose paper. Thus, the silane coupling treatment
of cellulose paper is a facile and useful technique for introducing
functional groups and improving physical strength and
hydrophobicity.
Immobilization of lipase on methacrylate-modified paper
Immobilization of lipase on cellulose paper was carried out by
a simple soaking treatment with an aqueous suspension of crude
lipase powders. A piece of original or methacrylate-modified
paper was immersed in a lipase solution with insoluble compo-
nents contained in crude lipase powders. Since methacrylate-
modified paper had some degree of hydrophobicity, it was
forcibly immersed in the suspension. Subsequently, the mixture
was stirred by using an orbital platform shaker. During the
soaking treatment for 12 h, the paper samples were physically
durable due to their acceptable wet strength. Insoluble compo-
nents contained in crude lipase powders may clog the paper pores
and disturb the immobilization of lipase. However, methacry-
late-modified paper achieved effective immobilization of lipase
even in the presence of the insoluble components; the immobi-
lization yield of lipase on methacrylate-modified paper reached
ca. 95%, while that on original paper was ca. 30%. The immo-
bilized lipase on the paper was thoroughly washed with phos-
phate buffer solution at a stirring rate of 150 rpm for 3 h to
remove unwanted insoluble components adsorbed onto the
Fig. 2 XRF spectra of (a) original cellulose paper and (b) cellulose paper
treated with 3-(trimethoxysilyl)propyl methacrylate.
Fig. 3 SEM images of (a) original paper and (b) methacrylate-modified
paper.
17,18
paper. The relatively hydrophobic nature of lipases
suggests
that lipases were effectively attached to the hydrophobic surfaces
of methacrylate-modified paper due to hydrophobic interaction.
The modification of cellulose paper with methacryloxy groups
therefore contributed to the effective immobilization of lipase.
between the OH groups of cellulose. In the wet condition,
however, water molecules intrude into cellulose paper and break
hydrogen bonds, resulting in a fatal decrease in the paper
strength. As shown in Table 1, methacrylate-modified paper had
a higher physical strength, including Young’s modulus and
tensile strength, than the original paper, regardless of dry or wet
state. It is notable that the wet tensile strength of methacrylate-
modified paper was more than 7 times higher than that of orig-
inal paper, leading to good practical utility even in water. This
possibly resulted from the formation of bridges between cellulose
fibers and silane coupling agents. In other words, silane coupling
agents acted as cross-linkers by providing both condensation
with the C–OH groups of cellulose and self-condensation
Fig. 4 Optical images and contact angles of water droplets on (a)
original paper and (b) methacrylate-modified paper.
Table 1 Physical properties of unmodified and methacrylate-modified cellulose paper
Young’s modulus/GPa
Tensile strength/MPa
a
a
Weight/mg
Thickness/mm
Dry
Wet
Dry
Wet
b
Unmodified paper
Methacrylate-modified paper
87.8 ꢃ 0.8
201.3 ꢃ 4.9
223.3 ꢃ 1.7
0.37 ꢃ 0.06
0.56 ꢃ 0.04
0.04 ꢃ 0.01
0.17 ꢃ 0.02
6.71 ꢃ 0.29
10.10 ꢃ 0.42
0.76 ꢃ 0.09
5.79 ꢃ 0.53
Dry condition: 23 C, 50% relative humidity. Unmodified paper was prepared by the similar procedure as methacrylate-modified paper in the absence
115.2 ꢃ 2.8
a
ꢁ
b
of any silane coupling agent, and the data were referred to ref. 37.
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Nonaqueous transesterification by immobilized lipase on
methacrylate-modified paper
In recent years, lipase-catalyzed transesterification reactions in
nonaqueous media have attracted much attention for use in
production of various useful chemicals through organic
3,51,52
synthesis.
For this reason, the immobilized lipase on meth-
acrylate-modified paper was used in the nonaqueous trans-
esterification reaction between 1-phenylethanol and vinyl acetate
to produce 1-phenylethylacetate (Scheme 1). Fig. 5 and Table 2
show enzymatic activities in batch-mode nonaqueous trans-
esterification of free lipase and the immobilized lipase on original
paper and methacrylate-modified paper. Free lipase without
supporting materials had a low specific activity (0.63). Although
isopropyl ether used as a reaction solvent in this study has been
reported to be relatively harmless for lipase activity as compared
with polar organic solvents such as methanol, ethylacetate
Fig. 5 Time course of GC yield of 1-phenylethylacetate in batch mode:
free lipase (triangles) and immobilized lipase on original paper (squares)
ꢁ
and methacrylate-modified paper (circles). Reaction temperature: 23 C.
Stirring rate: 150 rpm.
53,54
and pyridine,
free lipase would be easily aggregated even in
isopropyl ether, leading to a poor specific activity. In the case of
the immobilized lipase on methacrylate-modified paper,
however, specific activity was significantly improved at 25.3,
which is ca. 40-fold higher than that of free lipase. Immobilized
lipase on original paper had poor specific activity (0.31) and
methacrylate-modified paper without lipase had no catalytic
activity for this reaction. Many researchers have reported that
hydrophobic solid supports allow effective interaction with the
large hydrophobic pocket surrounding the catalytic site of
handle. In practice, the immobilized lipase on methacrylate-
modified paper was easily recoverable after the performance test
and was reusable without significant decrease in a specific activity
(
Table 2). Thus, the immobilization of lipase on methacrylate-
modified paper led to the improvement of activity and reusability
for nonaqueous transesterification, without notable loss of
enantioselectivity.
15,17–20
lipases,
leading to hyperactivation of lipases via interfacial
Flow reaction through an interconnected porous microstructure
of immobilized lipase on methacrylate-modified paper
activation, i.e. the formation of a suitable open structure for
17,18
efficient contact with substrates.
Thus, these results suggest
Flow-type catalytic reactions have received increasing attention
for the continuous production of target substances. In accor-
dance with this trend, there is growing interest in microstructured
that the hydrophobicity of the methacrylate-modified paper
support (Fig. 4) contributes to the improvement of lipase activity
for nonaqueous transesterification. In other words, lipase
activity improved by the immobilization on methacrylate-
modified paper due to hyperactivation of lipases and suppression
of aggregate formation, leading to a large difference of activities
between free and immobilized lipase. The specific activity of the
immobilized lipase on methacrylate-modified paper (25.3)
was comparable with those of the immobilized lipase on other
5
7–61
22,23,29
reactors
11,62
foams,
such as microchannels
and monolith
which contain micrometre-scale open paths for fluids,
because they allow fast mixing of reactants through small pores,
leading to high reaction efficiency. In this study, the immobilized
lipase on methacrylate-modified paper was tested in flow mode
by taking advantage of the paper-specific interconnected porous
microstructure (Fig. 3). Fig. 7 compares the productivities of
10
supporting materials previously reported, such as methacry-
late-modified porous ceramics (37.2), diatomaceous earth (7.6),
glass beads (0.8) and synthetic resins (0.4). Fig. 6 shows an
enantioselectivity of the immobilized lipase on methacrylate-
modified paper. When (R)-1-phenylethanol was used as
a substrate, the specific activity reached 28.2. In the case of (S)-1-
phenylethanol, however, the transesterification reaction did not
progress at all. These results indicate an acceptable enantiose-
lectivity of the immobilized lipase on methacrylate-modified
paper for (R)-1-phenylethanol, in common with other immobi-
1
-phenylethylacetate in batch and flow modes. The immobilized
lipase on methacrylate-modified paper demonstrated acceptable
permeability of the substrate solution within a flow rate of 2.0 ml
ꢀ1
min possibly due to its highly porous structure, and was more
effective in flow mode than in batch mode, with a maximum
ꢀ1
productivity recorded at a flow rate of 1.0 ml min that was ca.
-fold higher than that in batch mode at a stirring rate of 300
2
rpm. In the case of batch processes, which were conducted in
a closed glass vial (40 mm in diameter, 75 mm in height), the
millimetre-scale diffusion length of the reactants possibly results
in a relatively low productivity because the accessibility of the
reactants to the immobilized lipase on the paper would be
insufficient, even at a stirring rate of 300 rpm. On the other hand,
the flow reaction proceeded through the interconnected micro-
metre-scale pores of the paper. Such short diffusion paths inside
the paper would provide fast mixing of the reactants and effective
transport of the reactants to the catalytically active sites of
lipases, leading to a high productivity. Cellulose is highly stable
to most solvents and has both a hydrophilic and a lipophilic
10,55,56
lized lipases previously reported.
The advantages of paper
as a support over conventional materials include its excellent
practical utility, in that it is lightweight, flexible and easy to
Scheme 1 Lipase-catalyzed nonaqueous transesterification between
63–65
1-phenylethanol and vinyl acetate to produce 1-phenylethylacetate.
nature.
These unique properties are advantageous for use in
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Table 2 Enzymatic activities in batch-mode nonaqueous transesterification of free lipase and lipase immobilized on original paper and on methac-
ꢁ
rylate-modified cellulose paper. Reaction temperature: 23 C. Stirring rate: 150 rpm
a
U /mmol product per min
Supporting material
Lipase protein content/mg
Specific activity/U per mg protein
None
Original paper
Methacrylate-modified paper
56.0
16.2
52.6
0.035
0.005
1.33
0.63
0.31
25.3(22.5 )
b
a
b
One unit of lipase activity (U) was defined as the amount of enzyme required to produce 1 mmol of 1-phenylethylacetate per minute. Specific activity
at the third cycle.
paper-specific porous microstructure allowed higher productivity
for continuous flow reaction systems than in batch reaction,
possibly through provision of favorable diffusion paths for the
reactants. The additional advantages of cellulose paper over
other support materials may include sustainability and high mass
productivity, while the silane coupling technique for the intro-
duction of functional groups into cellulose paper is facile and
versatile. Thus, chemically modified cellulose paper is a green
support material that provides both excellent practical utility and
favorable reaction fields for enzymes and other catalysts.
Acknowledgements
Fig. 6 Enantioselectivity of immobilized lipase on methacrylate-modi-
ꢁ
fied paper. Reaction temperature: 23 C. Stirring rate: 150 rpm.
This research was supported by a Research Fellowship for
Young Scientists from the Japan Society for the Promotion of
Science (H. K.).
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