Novel immobilization method of enzymes using a hydrophilic polymer support

Article abstract of DOI:10.1039/b609335c

A novel immobilization of an enzyme with a hydrophilic polymer support in organic solvents has been developed utilizing the "polymer-incarcerated (PI) method", which has been used to immobilize metal catalysts; the kinetic resolution of secondary alcohols

Full text of DOI:10.1039/b609335c

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Novel immobilization method of enzymes using a hydrophilic polymer
support{
Juta Kobayashi, Yuichiro Mori and Shu¯ Kobayashi*
Received (in Cambridge, UK) 30th June 2006, Accepted 10th August 2006
First published as an Advance Article on the web 30th August 2006
DOI: 10.1039/b609335c
A novel immobilization of an enzyme with a hydrophilic
polymer support in organic solvents has been developed
utilizing the ‘‘polymer-incarcerated (PI) method’’, which has
been used to immobilize metal catalysts; the kinetic resolution
of secondary alcohols was found to proceed more smoothly
using immobilized lipases (CALB) than free lipases.
Enzymes, which catalyze a variety of reactions and play an
indispensable role in living systems, have many specific features,
such as highly controlled regio-, stereo- and substrate-specificity. In
addition, enzymatic reactions generally proceed under very mild
conditions. Therefore, enzymes have found wide application in the
pharmaceutical sciences, chemical synthesis and biotechnology,
etc. Even though a large number of efficient chemical reactions
have been well developed, enzymes still play a significant role in
providing many useful chemical products.1
Scheme 1 Preparation of polymer-immobilized lipase.
enzymes has been widely conducted in aqueous phases, the
corresponding immobilization procedure conducted in organic
solvents is much less well known. Herein, we describe a new
immobilization method for lipase in an organic phase and disclose
our results of using the immobilized lipase for the kinetic resolution
of secondary alcohols, a well known and representative reaction
that is promoted by lipases.
However, the use of enzymes is subject to limitations and
drawbacks in some cases. For example, generally speaking,
enzymes are relatively expensive, and therefore discarding them
after a single use is not economical. Another problem is their
general instability towards heat, organic solvents, acids or bases,
etc. One method to circumvent these issues is to immobilize the
enzyme on a suitable supporting medium. Not only does this
facilitate easier recovery and reuse of the enzyme, but in many
cases, immobilized enzymes show a higher stability than free
species. In recent years, a variety of methods for the immobiliza-
tion of enzymes, such as attachment to prefabricated carriers (by
covalent or ionic binding, adsorption, etc.) or cross-linkage and
entrapment (encapsulation), have been developed.2 Among these,
entrapment—where covalent bonding, which may alter the enzyme
structure and cause loss of activity, is avoided—is an effective
method for immobilization, While several types of entrapped
enzymes in liposomes, micelles and membranes, etc. have been
reported,3 we have developed the ‘‘Polymer-Incarcerated (PI)’’
method4 in our laboratory for immobilizing a variety of metal
catalysts. A representative example of this method is PI-Pd, which
exhibits excellent catalytic properties for typical reactions mediated
by palladium, such as the Heck reaction, allylic substitution and
hydrogenation, etc.4a,b Recently, this immobilization method has
been successfully applied to microchannel reactors, providing
efficient systems for hydrogenation.5 Whilst the immobilization of
The immobilization procedure is depicted in Scheme 1. Lipase B
from Candida antarctica (CALB)6 was selected as the candidate
enzyme and copolymer 1 was chosen as the polymer support. This
copolymer contains hydrophilic moieties such as tetraethylene
glycol (TEG) and glycidol, and thus hydrophilic interactions
between the copolymer and the liquid lipase were expected. First,
copolymer 1 was dissolved in CH2Cl2, and then the liquid lipase
was dispersed into the solution by stirring vigorously. After the
lipase had been dispersed, hexane was slowly added. This caused
coacervation to occur, forming a precipitate with lipase particles
inside the polymer. At this stage, most of the lipase was in the
polymer phase. The supernatant was removed by decantation, and
then cross-linking was conducted using amine 2 as a cross-linking
Table 1 Reuse of the catalyst
Yield,a ee (recovered alcohol) (%)
1st
2nd
3rd
4th
5th
42, . 99
(42, 99)
42, . 99
(42, 99)
44, . 99
(42, 99)
45, . 99
(44, . 99)
43, . 99
(43, . 99)
Graduate School of Pharmaceutical Sciences, The University of Tokyo,
The HFRE Division, ERATO, Japan Science and Technology Agency
(JST), Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
30, . 99b
8, . 99b
—
—
—
a
b
Isolated yield. Free liquid lipase (100 mg) was used. The yield
and ee of the recovered alcohol were not determined.
E-mail: skobayas@mol.f.u-tokyo.ac.jp; Fax: +81 3 5684 0634
{ Electronic supplementary information (ESI) available: Experimental
section. See DOI: 10.1039/b609335c
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Chem. Commun., 2006, 4227–4229 | 4227

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Table 2 Kinetic resolution of secondary alcohols
Ester
Ester
Ester
Substrate
Yielda
17
ee (%)
Substrate
Yielda
42
ee (%)
Substrate
Yielda
40
ee (%)
. 99
. 99
. 99
46
47
. 99
. 99
44
45
. 99
3
n.d.b
98
43
98
a
b
Isolated yield. Not determined.
agent at 60 uC in hexane. After 12 h, the supernatant was again
decanted off and the residue washed with organic solvents such as
dichloromethane, diethyl ether or ethyl acetate. The residue was
then dried in vacuo, giving a polymer-immobilized lipase (242 mg
for 1 reaction). The loading level of the lipase was estimated
indirectly as follows: First, the organic washing solutions used
during the immobilization process were combined, extracted with
water and the amount of protein in the water phase determined by
Lowry’s method.7 Next, the loading of the immobilized lipase was
calculated by subtracting the amount of protein found in the
aqueous phase from the total amount of protein initially used.
Using this method, the loading was estimated to be 72.5 mg
protein g21, indicating that more than 90% of the lipase had been
immobilized onto the polymer.
the case of the immobilized lipase. The second use of free lipase
gave less acetylated product, implying that lipase was deactivated
by organic solvents and that protection of an enzyme by a polymer
support might take effect in the case of an immobilized lipase.
Encouraged by these results, we next examined other substrates
(Table 2). The reaction proceeded with various kinds of secondary
alcohols. For example, those having naphthyl groups, several
substituted benzene groups or cyclic alcohols gave good resolution,
with the exception of 1-phenyl-1-propanol. The reaction rate
depended on the substrate: some were complete within several
hours while others required longer reaction times.
In summary, a new immobilization method of lipase in organic
phases has been developed utilizing a hydrophilic polymer support.
The immobilization method is based on the PI method, which has
previously been used to immobilize metal catalysts. The immobi-
lized lipase exhibits high enzymatic activity, and efficient kinetic
resolution of secondary alcohols has been attained. Recovery and
reuse of the immobilized lipase is possible, implying a higher
stability against organic solvents than free lipase. Further
applications of the immobilization method to other enzymes
and/or microchannel reactors are under investigation.
Using the polymer-immobilized lipase obtained, we next
examined the kinetic resolution of secondary alcohols. Racemic
1-phenylethyl alcohol was used as a starting substrate to assess the
activity and reusability of the immobilised enzyme. The reaction
was conducted in diethyl ether at 25 uC for 24 h using vinyl acetate
as an acetylating agent. The results are shown in Table 1. During
the reaction, the immobilized lipase dispersed into the reaction
mixture, and the reaction proceeded to give both the product and
the recovered alcohol in good yields with excellent enantioselec-
tivity. After the reaction was complete, the organic liquid phase
was collected by decantation, the residue washed with organic
solvents, and the remaining immobilized polymer dried and used
for the next trial. In this way, it was found that the polymer-
immobilized lipase could be recovered and reused at least five
times without loss of activity. As a control experiment, the free
lipase before immobilization was used and the reaction conducted
under the identical conditions. This reaction proceeded sluggishly,
which might be ascribed to insufficient contact between the lipase
and the substrate due to the liquid lipase remaining at the bottom
of the flask during the reaction. After decantation and washing
with organic solvents, the lipase was recovered and reused, as in
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