Facile preparation of an enzyme-immobilized microreactor using a cross-linking enzyme membrane on a microchannel surface

Article abstract of DOI:10.1002/adsc.200606224

The enzyme microreactor has considerable potential for use in biotechnological syntheses and analytical studies. Simplifying the procedure of enzyme immobilization in a microreactor is attractive, and it is achievable by utilizing enzyme immobilization techniques and taking advantage of the characteristics of microfluidics. We previously developed a facile and inexpensive preparation method for an enzyme-immobilized microreactor. The immobilization of enzymes can be achieved by the formation of an enzyme-polymeric membrane on the inner wall of the microchannel through cross-linking polymerization in a laminar flow. However, this method is unsuitable for use in conjunction with electronegative enzymes. Therefore, a novel preparation method using poly-L-lysine [poly(Lys)] as a booster and an adjunct for the effective polymerization of electro-negative enzymes was developed in this study. Using aminoacylase as a model for an electronegative enzyme, the reaction conditions for the enzyme-cross-linked aggregation were optimized. On the basis of the determined conditions, an acylase-immobilized tubing microreactor was successfully prepared by cross-linking polymerization in a concentric laminar flow. The resulting microreactor showed a higher stability against heat and organic solvents compared to those of the free enzyme. The developed method using poly(Lys) was applicable to various enzymes with low isoelectric points, suggesting that this microreactor preparation utilizing a cross-linked enzyme in a laminar flow could be expanded to microreactors in which a broad range of functional proteins are employed.

Full text of DOI:10.1002/adsc.200606224

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DOI: 10.1002/adsc.200606224
Facile Preparation of an Enzyme-Immobilized Microreactor using
a Cross-Linking Enzyme Membrane on a Microchannel Surface
Takeshi Honda,a Masaya Miyazaki,a,b,* Hiroyuki Nakamura,a
and Hideaki Maedaa,b,*
a
Nanotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 807-1
Shuku, Tosu, Saga 841-0052, Japan
Phone: (+81)-942-81-3676, Fax: (+81)-942-81-3657; e-mail: m.miyazaki@aist.go.jp or maeda-h@aist.go.jp
Department of Molecular and Material Science, Interdisciplinary Graduate School of Engineering Sciences, Kyushu
b
University, 6–1 Kasuga Koen, Kasuga, Fukuoka 816-8580, Japan
Phone/Fax: (+81)-92-583-8891
Received: May 19, 2006; Accepted: August 15, 2006
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: The enzyme microreactor has considerable aminoacylase as a model for an electronegative
potential for use in biotechnological syntheses and enzyme, the reaction conditions for the enzyme-
analytical studies. Simplifying the procedure of cross-linked aggregation were optimized. On the
enzyme immobilization in a microreactor is attrac- basis of the determined conditions, an acylase-immo-
tive, and it is achievable by utilizing enzyme immobi- bilized tubing microreactor was successfully prepared
lization techniques and taking advantage of the char- by cross-linking polymerization in a concentric lami-
acteristics of microfluidics. We previously developed nar flow. The resulting microreactor showed a higher
a facile and inexpensive preparation method for an stability against heat and organic solvents compared
enzyme-immobilized microreactor. The immobiliza- to those of the free enzyme. The developed method
tion of enzymes can be achieved by the formation of using poly(Lys) was applicable to various enzymes
G
an enzyme-polymeric membrane on the inner wall of with low isoelectric points, suggesting that this micro-
the microchannel through cross-linking polymeri- reactor preparation utilizing a cross-linked enzyme
zation in a laminar flow. However, this method is un- in a laminar flow could be expanded to microreac-
suitable for use in conjunction with electronegative tors in which a broad range of functional proteins
enzymes. Therefore, a novel preparation method are employed.
using poly-l-lysine [poly(Lys)] as a booster and an
E
adjunct for the effective polymerization of electro- Keywords: cross-linked enzyme aggregation; enzyme
negative enzymes was developed in this study. Using microreactor; immobilization; surface modification
Introduction
fields, especially for high-throughput biotechnological
syntheses and bioanalyses.[3] In such microreactor
Microreactors are potentially powerful tools in the preparations, nanostructure fabrication and chemical
fields of chemistry and biotechnology.[1] The excellent modification of the microchannel are useful technolo-
performance of microreaction systems is achieved by gies.[4] We have also developed a method for immobi-
taking advantage of microchannel system features lizing enzymes on a glass-capillary surface using a
such as rapid heat and mass transfer, and their larger chemical modification containing sol-gel techniques.[5]
surface/interface area. These attractive features are However, these methods require high-level techni-
favorable for conducting catalytic reactions through ques and multi-step procedures, leading to low cost
catalysts that are immobilized on the reactor.[2] En- performance. More simple methods using carriers
zymes are useful catalysts for the production of cer- with immobilized enzymes have also been proposed.
tain types of chemicals and analysis, which are fre- The carriers were constructed by forming monoliths
quently applied to various reactors. There have been of a sol-gel and a polymer, and simply incorporating
demands for enzymatic microreaction devices includ- commercial beads and membranes in a microchan-
ing enzyme-immobilized microreactors in several nel.[6] These structures are often responsible for the
Adv. Synth. Catal. 2006, 348, 2163 – 2171
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Results
The Effect of a Cationic Polymer on CLEA
Formation of an Electronegative Protein
The efficient formation of a CLEA is essential for the
successful preparation of a CEM. We first explored
an efficient method in a batchwise system. Glutaral-
dehyde (GA) and paraformaldehyde (PA) were used
as cross-linkers, as previously described.[8] When
mixing the cross-linker and enzyme, CLEAs for elec-
tropositive enzymes such as chymotrypsin and trypsin,
which contain a high number of lysine residues, are
readily formed. On the other hand, no CLEAs were
observed in the case of electronegative enzymes. The
relatively low content of lysine residues in these en-
zymes would cause a low degree of cross-linking,
leading to inefficient aggregation. To achieve effective
Scheme 1. Reaction of enzyme amino groups and/or poly-
(Lys) with aldehyde groups of the cross-linker.
generation of high pressure, which is unfavorable for cross-linking polymerization, we proposed the close
microsynthesis and -analysis systems with multiple packing of electronegative enzyme molecules by
processes based on highly-controlled microfluids,[7] enzyme-trapping using a cationic polymer matrix
and are energy-intensive to operate. Therefore, in our prior to the cross-linking reaction.[9] The polymer
previous study, we designed an enzyme-immobilized matrix, possessing multiple basic functional groups,
microreactor which was simply and inexpensively pre- and electronegative enzymes will be adsorbed by elec-
pared and does not require high pressure.[8] In our mi- trostatic interactions, and form an enzyme-enzyme
croreactor process, a substrate membrane covers the complex bridged by the matrix. Actually, poly(Lys)
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internal surface of the microchannel. The cylindrical immobilized on a carrier is frequently utilized to cap-
membrane is composed of the cross-linked polymer- ture electronegative proteins.[11,12] In this study, we
ized enzyme product. The hollow structure of this re- used poly
actor prevents high pressure, compared to carrier-fill- gregate of the electronegative enzyme.
ing microreactors.[6,8] In addition, this carrier-free
The effect of poly(Lys) on CLEA formation was
A
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method would be expected to have the advantage of compared between chymotrypsin and aminoacylase
an absence of interactions between the enzyme and (hereafter referred to as acylase) as electropositive
the carrier material, which often leads to enzyme in- and electronegative model enzymes, respectively. The
activation.[9] This bottom-up method is suitable for turbidity in the reaction mixture increases with in-
the formation of miniature structures, and has recent- creasing amounts of insoluble aggregates. Thus, the
ly been utilized in various nanotechnological applica- progress of CLEA formation could be evaluated by
tions such as enzyme-polymerized nanoparticles.[10] the turbidity of the solution, as estimated by measur-
The enzyme-polymerized membrane was based on a ing the absorbance at 630 nm. As shown in Figure 1,
cross-linked enzyme aggregate (CLEA)[9] formed the cross-linker consisted of GA (0.25%, v/v) and PA
using a cross-linker with aldehyde groups which react (4%, v/v) agglutinated chymotrypsin, but not acylase.
with amino groups of the enzyme (Scheme 1). There- No turbidity was observed when concentrated acylase
fore, CLEAs could be not obtained for electronega- (5-fold; 100 mgmLÀ1) and/or cross-linker (5-fold;
tive enzymes due to a lack of reactive amino groups 1.25% GA and 20% PA) was used (data not shown).
(relatively lower number of lysine residues). In the In contrast, the addition of the cross-linker into the
present study, to demonstrate the applicability of the mixture of acylase and poly
CLEA-based enzyme-microreactor (CEM) method a remarkable increase in the turbidity of the reaction
for various enzymes, we developed a novel method solution. When mixing acylase and poly(Lys) without
for preparing a CEM that is applicable to electroneg- cross-linker, in which an acylase-poly(Lys) complex
ative enzymes utilizing poly-l-lysine [poly(Lys)]. The would be formed as a result of electrostatic interac-
successful preparation was found to realize an effi- tions, the solution contained negligible suspended
cient and fast CLEA formation in electronegative en- solids (Figure 1). Poly(Lys) and the cross-linker mix-
zymes by using poly(Lys). ture without acylase, in which the cross-linking reac-
tion would occur between poly(Lys) molecules,
showed no turbidity (Figure 1). These results can be
attributed to the high hydrophilicity of poly(Lys)
which enables each polymeric complex to prevent in-
(Lys) (Mn =20 kDa) led to
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Facile Preparation of an Enzyme-Immobilized Microreactor
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solubilization. It is clear that the turbidity of the acy-
lase-poly
concerted reaction of the three reagents. A reaction
using lysine instead of poly(Lys) showed no evidence
of CLEA formation, indicating the importance of the
polymeric structure of poly(Lys) for effective CLEA
(Lys)-cross-linker mixture is a result of the
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formation. The addition of lysine to a mixture of chy-
motrypsin-cross-linker greatly reduced the turbidity
of the reaction mixture. This deleterious effect might
be due to competition between amino groups of the
enzyme and lysine, which possess little cross-linking
ability.
In order to achieve broad applicability for CEM for
various enzymes, we examined whether CLEAs were
formed using poly(Lys), using several industrially
U
useful enzymes with distinct isoelectric points. Table 1
shows the relationship between the isoelectric points
of each enzyme and CLEA formation. Electropositive
enzymes, including chymotrypsin, cucumisin, papain
Figure 1. Effect of poly(Lys) on CLEA formation for elec-
A
and trypsin easily formed a CLEA without poly
In other enzymes, with low isoelectric points (pI<6),
the addition of poly(Lys) permitted successful CLEA
formation. These results indicate that poly(Lys) has a
A
tropositive chymotrypsin and electronegative acylase. Each
bar shows the turbidity of the suspensions after incubation
for 60 min at 48C. A high turbidity means effective forma-
tion of the CLEA. The addition of each solution is de-
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scribed as “+” for each reagent and “À
reagents. “(+)” indicates the addition of lysine instead of
poly(Lys).
” for buffer without
strong potential for CLEA formation for a broad
range of electronegative enzymes.
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Preparation of the Acylase-CEM
Table 1. CLEA formation in several enzymes of distinct iso-
electric points.
Effective CLEA formation in an electronegative
enzyme was achieved utilizing poly(Lys). Thus, we ap-
A
pI[a]
CLEA[b]
formed with poly(Lys)
plied this technology to a CEM preparation. The elec-
tronegative enzyme used was an acylase, which is very
interesting from a biotechnology standpoint, because
of their relevance in the production of enantiopure
amino acids from N-acyl derivatives.[13,14] In order to
achieve the effective preparation of acylase-CEM
with a high enzymatic activity, we optimized the
CLEA preparation conditions such as the concentra-
tion of cross-linker, the molecular weight of poly-
Enzyme
Alkaline phosphatase
l-Aminoacylase
d-Aminoacylase
b-d-Galactosidase
a-Chymotrypsin
Cucumisin
5.9
4.0
5.2
4.6
8.6
10.4
5.0
4.5
8.8
1.0
9.4
5.3
10.5
5.2
3.7
G
formed with poly(Lys)
formed with poly(Lys)
formed with poly(Lys)
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formed without poly
formed without poly
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Esterase
formed with poly
formed with poly
formed without poly
formed with poly(Lys)
formed without poly(Lys)
formed with poly(Lys)
formed without poly(Lys)
formed with poly(Lys)
formed with poly(Lys)
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Lipase, Candida rugosa
Papain
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A
N
Pepsin A[c]
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ing Information, Figures S1 and S2). The acylase-
CEM was then prepared at these optimal conditions.
The following reagents were used: acylase
Subtilisin A
Thermolysin
Trypsin
Tyrosinase
V8 protease
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(20 mgmLÀ1), poly(Lys) (Mn =50 kDa, 10 mgmLÀ1),
T
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and cross-linker corresponding to GA (0.03%) and
PA (0.5%). The acylase solution was mixed with poly-
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[a]
Each value of isoelectric point (pI) was determined by
isoelectric focusing in previous studies.[31–43] A pI value of
b-d-galactosidase was obtained from manufacturerꢁs data
sheet.
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sive preparation, a commercially available PTFE tube
was employed as a substrate for the CEM (hereafter
described as CEM-tube).[8] The CLEA-based mem-
brane in the CEM-tube was formed utilizing a charac-
teristic microflow, namely, a concentric laminar flow
consisting of two solutions of the cross-linker and acy-
[b]
[c]
Each CLEA preparation was performed by mixing
enzyme (20 mgmLÀ1) and cross-linker (0.25% GA and
4% PA) in the presence or absence of poly
50 kDa, 10 mgmLÀ1).
A
For pepsin, lower pH values (pH 5) was used because
pepsin is rapidly and irreversibly denaturated at alkaline
values, but is stable until pH 5.5.[44]
lase-poly
A
consisting of axle dual micropipes, as shown in
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Figure 2. Schematic illustration of the procedure used to prepare an acylase-CEM. The cross-linking polymerization was per-
formed in a concentric laminar flow. A silica capillary was fitted to the outer diameter of the T-shape connector by attaching
to a PTFE tube using heat-shrink tubing. The capillary was set in the connector located at the concentric position of the
CEM tube. The cross-linker solution was supplied to the substrate PTFE tube through the silica capillary, corresponding to a
central stream in the concentric laminar flow. A solution of acylase-poly
T-shape connector, and formed an outer stream of the laminar flow.
A
lindrical membrane was shrunk by drying, and the
thickness of the obtained membrane was 35–60 mm.
When a suspension of CLEA prepared in a batchwise
system was passed through a PTFE tube, no mem-
brane was observed on the inner wall (data not
shown). This result indicates that polymerization in a
microfluid enables the enzyme-polymer product to
adopt a membrane structure on the inner wall of the
CEM tube. The amount of acylase, estimated by a
quantitative amino acid analysis as described previ-
ously,[8] was 3.25Æ0.17 nmol per acylase-CEM.
Figure 3. CCD images of a) cylindrical enzyme-membrane
(dry state) exposed from the PTFE tube, which forms on
the inner wall of the tube and b) a sectional view of the ob-
tained CEM.
Performance of the Acylase-CEM
Figure 2.[15] A stable laminar flow is important for the
successful formation of a CLEA-based membrane on The enzymatic performance of the obtained acylase-
the internal surface of a CEM-tube,[8] and appropriate CEM was evaluated using the hydrolysis of acetyl-
flow rates should be selected for each solution. We d,l-phenylalanine (Ac-d,l-Phe) at various conditions.
set the pumping rate of the cross-linker (correspond- The CEM was cut off at both ends to a length of
ing to a central stream) to a value 12.5 times higher 12.2 cm, corresponding to 24 mL of inner volume. The
than that of the acylase-poly(Lys) mixture (outer hydrolysis at 4 mM of substrate was complete at this
A
stream). The formation of a concentric laminar flow flow rate (1 mL minÀ1) (Figure 4). The Michaelis–
was confirmed by confocal laser scanning microscopy Menten constant (Km) and maximal velocity (Vmax),
(see Supporting Information, Figure S3). Calculating calculated from a Lineweaver–Burke plot, were
from the two distinct pumping rates and microfluidic 4.58 mM and 1.10 mMminÀ1, respectively. All prod-
volumes estimated from the confocal data, average ucts showed high enantiopurities (99.3Æ0.8%) com-
linear velocities of the central and outer streams were pared to those obtained in a batch process (99.2Æ
187.6 mmminÀ1 and 6.4 mmminÀ1, respectively. In the 0.4%). When treating 100 mM substrate at a flow
CEM-tube, the residence time of the central stream rate of 4 mL minÀ1, the highest amount of l-Phe pro-
was much shorter (0.6 min) than that of the outer duction was 45 nmol per minute. Under similar reac-
stream (39.1 min). According to the kinetics of CLEA tion conditions, the Km and Vmax values for the free
formation in a batch (see Supporting Information, acylase were estimated to be 2.33 mM and
Figure S4), this residence time for the acylase-poly- 4.69 mMminÀ1, respectively. The Vmax value was
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The resulting CEM was washed with distilled acylase. These results suggest that chemical modifica-
water, dried and the enzyme-membrane observed. tion with the cross-linker somewhat suppressed the
Figure 3 shows CCD images of the cylindrical hydrolytic ability of the acylase.
enzyme-membrane (dry state) exposed from the
PTFE tube and a sectional view of the CEM. The cy- lase-poly
When a PTFE tube was passed through the acy-
(Lys) solution without a cross-linker, no en-
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