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Chemistry Letters Vol.38, No.3 (2009)
An Immobilized Lipase Microfluidic Reactor for Enantioselective Hydrolysis of Ester
Yan Gao, Runtao Zhong,1;2 Jianhua Qin, and Bingcheng Lin
Ã1
1
;2
Ã1
1
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, P. R. China
2
Graduate School of the Chinese Academy of Sciences, 19A Yuquanlu, Beijing 100049, P. R. China
(Received December 18, 2008; CL-081192; E-mail: bclin@dicp.ac.cn)
Enantioselective enzymatic hydrolysis of racemic mixture
is an effective method to obtain pure enantiomer. In this report,
an immobilized lipase microfluidic reactor was fabricated and
applied to enantioselective hydrolysis of racemic 1-phenylethyl
acetate to obtain (R)-1-phenylethanol. This immobilized lipase
microfluidic reactor consumed trace amount of enzyme, showed
identical performance compared to free enzyme in batch, and
good stability that can be recycled for at least eight times without
obvious loss of activity.
Production of chiral precursors or intermediates is now in
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high demand in the pharmaceutical and agricultural industry.
Figure 1. Immobilized lipase microfluidic reactor. (a) Sche-
matic illustrating the reaction of BCL catalytic hydrolysis of
rac-1-phenylethyl acetate, (b) setup of the microfluidic reactor
system, (c) photograph of the microfluidic reactor.
Great efforts have been made to develop different methods
to produce chiral compounds. Among various methods for ob-
taining chiral compounds, enzyme-catalyzed enantioselective
resolution has been considered to be useful, because enzymes
show high catalytic efficiency under mild conditions, and high
channel (200 mm wide, 25 mm deep) was fabricated in a 3 cm Â
2:5 cm  2:3 mm glass chip.
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chemo-, regio-, and enantioselectivities. Generally, enzymes
can be used in free or immobilized modes. Compared with a free
enzyme in solution, enzyme immobilized on the surface of
a suitable carrier material is more stable and convenient for
reuse.4
Compared with a conventional batch reactor, microfluidic
reactors are more effective in control of reaction condition, such
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as temperature and pH. For the model reaction used in this
work, the reaction medium is usually maintained near neu-
,5
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1,12
Microfluidic reactors are a new reaction platform which are
characterized by efficient heat transfer and mass transport, large
surface-to-volume ratio and microlitre-scale consumption of
reagent and sample, and have shown advantages such as speed,
throughput, and controllability in the field of chemical synthesis
in some cases.6 Compare with glass capillary, a microfluidic
reactor is more flexible to design and easy to be integrated with
other operation units such as micro-mixer, micro-extractor, mi-
cro-heater, etc. Recently, free enzyme-catalyzed asymmetric re-
action and separation of (S)-ibuprofen on microfluidic reactors
tral,
because pH fluctuation could affect the efficiency of
hydrolysis by affecting the activity of enzyme. In conventional
batch reaction, to maintain the pH of the reaction medium,
low concentration of NaOH was added to the reaction medium
by an automatic titration to decrease the effect of the acetic acid
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produced. By using the immobilized enzymatic microfluidic
reactor, the products and unreacted reactant were continuously
pushed out from the reaction channel, the following reactant
entered the reaction channel was little affected by the acetic acid
produced in prophase.
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has been carried out. To our knowledge, however, immobi-
lized enzyme-catalyzed racemate resolution in microfluidic re-
actors has not been reported yet. Here, we fabricated an immo-
bilized enzymatic microfluidic reactor made of glass, and ap-
plied it to enantioselective resolution of ester. Immobilized
lipase from Burkholderia cepacia (BCL) catalytic enantioselec-
tive hydrolysis of racemic 1-phenylethyl acetate was used as
a model reaction, seen in Figure 1a. In this reaction, the lipase
selectively hydrolyzes the (R)-1-phenylethyl acetate and pro-
duces (R)-1-phenylethanol, acetic acid and unreacted (S)-1-
phenylethyl acetate.
Lipase was covalently immobilized on the wall of micro-
fluidic reactor according to the method reported previously.13
Firstly, the channel was silanized by 10% (V/V) 3-aminopropyl-
trimethoxysilane (APTS) in methanol. Then 10% (V/V) gluta-
raldehyde dissolved in phosphate buffer (20 mM, pH 8.0) was in-
troduced into the channel for 3 h at room temperature. Finally,
enzyme solution was applied to the channel with low velocity
of flow for approximately 24 h. Then the reactor channel was
washed with phosphate buffer (20 mM, pH 7.4) and stored at
ꢀ
4 C until use. Here, glutaraldehyde not only connected with li-
pase, but also offered a space arm with five carbon atoms, which
may contributed to keep the reactivity of the lipase. The content
of the lipase immobilized on the wall of the microfluidic reactor
Figure 1b shows the setup of the microfluidic reactor system
used in this work. The modification of the microfluidic reactor
channel and the introduction of the substrate were performed
by a syringe pump (Harvard Apparatus, USA). Figure 1c dis-
plays the microfluidic reactor. The reactor was fabricated in
borosilicate glass following standard photolithographic and
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channel was measured using a micro-BCA method. Approxi-
mately 14 mg of lipase was covalently immobilized on the chan-
nel wall.
Diluted racemic ester (10 mM, 10 mL) was introduced into
the channel with different velocity of flow controlled by the
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wet chemical techniques. A 41-cm-long serpentine reaction
Copyright ꢀ 2009 The Chemical Society of Japan
Gao, Yan
