Synthesis of amino acid using a flow-type microreactor containing enzyme-mesoporous silica microsphere composites

Article abstract of DOI:10.1039/c3ra45315d

A flow-type microreactor containing composite materials of a theanine synthetase (glutaminase) and mesoporous silica with 23.6 nm pore diameter (SBA-15 microsphere) was developed for the continuous synthesis of l-theanine, a unique amino acid. Enzyme-immobilisation ability and enzymatic activity in the SBA-15 microsphere with large mesopores were higher than those of SBA-15 with a 5.4 nm pore diameter. Moreover, the glutaminase-SBA-15 microsphere composites displayed higher selectivity in theanine production than the free enzyme did in a batch experiment. A direct visualization of composites of fluorescently labelled glutaminase and SBA-15 microsphere immobilised in the flow channel of the microreactor by a combination of differential interference contrast and fluorescence microscopy revealed that the enzymes were uniformly dispersed throughout the mesoporous silica particles, because of the successful encapsulation of the enzyme. The enzyme-encapsulated microreactor exhibited a high conversion of l-glutamine to l-theanine with local control of the reaction temperature. In addition to this advantage of the microreaction system, the microreactor enabled the on-off regulation of enzymatic activity during continuous theanine synthesis by controlling the reaction temperature or the pH of the substrate solution.

Full text of DOI:10.1039/c3ra45315d

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Synthesis of amino acid using a flow-type
microreactor containing enzyme–mesoporous
silica microsphere composites†
Cite this: RSC Adv., 2014, 4, 9021
Shun-ichi Matsuura,* Manami Chiba, Emiko Tomon and Tatsuo Tsunoda
A flow-type microreactor containing composite materials of a theanine synthetase (glutaminase) and
mesoporous silica with 23.6 nm pore diameter (SBA-15 microsphere) was developed for the continuous
synthesis of L-theanine, a unique amino acid. Enzyme-immobilisation ability and enzymatic activity in the
SBA-15 microsphere with large mesopores were higher than those of SBA-15 with a 5.4 nm pore
diameter. Moreover, the glutaminase–SBA-15 microsphere composites displayed higher selectivity in
theanine production than the free enzyme did in a batch experiment. A direct visualization of
composites of fluorescently labelled glutaminase and SBA-15 microsphere immobilised in the flow
channel of the microreactor by a combination of differential interference contrast and fluorescence
microscopy revealed that the enzymes were uniformly dispersed throughout the mesoporous silica
particles, because of the successful encapsulation of the enzyme. The enzyme-encapsulated
microreactor exhibited a high conversion of L-glutamine to L-theanine with local control of the reaction
temperature. In addition to this advantage of the microreaction system, the microreactor enabled the
on-off regulation of enzymatic activity during continuous theanine synthesis by controlling the reaction
temperature or the pH of the substrate solution.
Received 24th September 2013
Accepted 21st January 2014
DOI: 10.1039/c3ra45315d
www.rsc.org/advances
Despite the remarkable progress in enzyme immobilisation
techniques, which rely on chemical cross-linking, physical
Introduction
The development of microscale reaction technologies, such as adsorption, or specic affinity bonding, current techniques can
microreactors, for use in the elds of enzyme and biological occasionally cause severe damage to the enzymes, e.g., by
engineering has recently attracted considerable attention blocking their active centres, inducing conformational
8
because of the many potential industrial applications for these changes, or forming aggregates. Although there are several
1,2
technologies. The advantage of using a puried, immobilised specic enzyme immobilisation techniques using affinity
enzyme-based microreactor for biocatalytic reactions over using bonds, such as those in the biotin–avidin system and the His-
9,10
macro scale systems with immobilised cells is that the enzyme- tag technique, which is based on the construction of a self-
immobilised microreactor facilitates the precise control of the assembled monolayer (SAM) of bifunctional linkers, these
reaction temperature to increase the reaction efficiency and techniques are still very expensive and time-consuming.
3–6
reduce the reaction time.
The selective and stable but Therefore, a new enzyme immobilisation technique that (a)
reversible immobilisation of an enzyme on a solid support does not cause inactivation of the enzyme, (b) performs a stable,
provides a reusable, continuous reaction system and offers a continuous reaction to yield the desired product, and (c) is
7
promising approach for use in industrial production processes.
relatively cost-efficient, is desired.
In order to achieve this goal, we have developed an enzyme-
encapsulated microreactor containing immobilised meso-
Research Center for Compact Chemical System, National Institute of Advanced porous silica capsules that have a hexagonally ordered pore
Industrial Science and Technology (AIST), 4-2-1 Nigatake, Miyagino-ku, Sendai
11–13
structure,
which acts as an encapsulation material for
9
+
†
83-8551, Japan. E-mail: matsuura-shunichi@aist.go.jp; Fax: +81-22-237-5215; Tel:
81-22-237-3039
14
delivery of proteins and for enzymes, which is known to
15,16
increase their stability and durability when exposed to heat,
Electronic supplementary information (ESI) available: Preparation procedure
17
18
19
chemicals, pH changes, and organic solvents. In addition
and characterisation of
a
PDMS/glass microreactor containing an
enzyme–mesoporous silica composite, photo and diagram of the experiment to improving the durability of the encapsulated enzyme, the
apparatus used for local heating, and tests for theanine production at various
effect of the pore diameter of 2D hexagonal mesoporous silica
reaction temperatures in batch reactions by free glutaminase and
glutaminase–SBA23.6 composite and at various residence times in microow
reactions using the glutaminase–mesoporous silica composites. See DOI:
20,21
on the catalytic activities of enzymes and proteins
efficiency of energy transfer between the encapsulated
and the
22–24
proteins
have also been studied. Pore-size optimisation is
1
0.1039/c3ra45315d
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vital for the appropriate expression of enzymatic activity in a synthesized from TEOS by using Pluronic P123 and mesitylene,
microreactor containing composites of enzyme and meso- as follows. To 120 mL of water was added 4 g of Pluronic P123,
porous silica material. Thus, the effect of pore diameter on the followed by 6.1 g of KCl; this mixture was stirred until trans-
adsorption capacity of silica and its reactivity toward enzymes lucent at room temperature. Then, 23.6 g of 12 N HCl and 3 g of
must be claried.
mesitylene were added to the above solution. This mixture was
To this end, we here rst investigated the ability of 2 types of stirred for 2 h at room temperature. Aer adding 8.5 g of TEOS
SBA-15 series of mesoporous silica (SBA5.4 and SBA23.6; pore to the above solution, the mixture was vigorously stirred for 10
ꢀ
diameters of 5.4 and 23.6 nm, respectively) to encapsulate a min at room temperature, and aged for 24 h at 35 C, and
ꢀ
recombinant glutaminase (theanine synthetase) and the subsequently aged for another 24 h at 130 C.
resulting enzymatic activity in the synthesis of a unique amino
acid found in tea, viz., g-glutamylethylamide (L-theanine), in times with 400 mL of distilled water at 80 C, and dried at 45 C.
batch experiments. Then, we conducted investigations of the The samples were calcined in air at a heating rate of 105 C h
on-chip encapsulation of glutaminase using the SBA-15 parti- and then held at 550 C for 10 h.
Next, the above solid products were ltered out, washed 3
ꢀ
ꢀ
ꢀ
ꢁ1
ꢀ
cles immobilised in
a ow-type microreactor. We also
conrmed the specic immobilisation of glutaminase on the
SBA-15 particles in the microow channel by direct visualiza-
tion of the immobilised glutaminase using optical microscopy,
and subsequently evaluated the performance of the enzyme
microreactor in the continuous synthesis of L-theanine.
Characterization of mesoporous silicas
In order to evaluate the pore diameters, pore volumes, and
specic surface areas of the calcined mesoporous silicas,
nitrogen adsorption and desorption measurements were carried
ꢀ
out at ꢁ196 C on BELSORP-max gas adsorption apparatus (BEL
Japan Inc., Osaka, Japan). The pore-size distributions were
Experimental
Silica source and templates
determined by analysing the adsorption branch via the Barrett–
28
Joyner–Halenda (BJH) method. The specic surface areas were
29
calculated by the Brunauer–Emmett–Teller (BET) method,
Tetraethyl orthosilicate (TEOS), as a silica source for meso-
porous silicas, was obtained from Wako Pure Chemical Indus-
using adsorption data ranging from P/P ¼ 0.05 to 0.25.
0
To conrm the morphology of the mesoporous silica parti-
cles, scanning electron microscope (SEM) observations were
carried out as follows. Scanning electron micrographs of the 2
mesoporous silicas (SBA5.4 and SBA23.6) were obtained using a
Miniscope (TM-1000; Hitachi High-Technologies Corp., Tokyo,
Japan) with an acceleration voltage of 15 kV.
tries
Ltd.
(Osaka,
Japan).
Triblock
copolymer,
poly(ethyleneglycol)-block-poly(propylene
glycol)-block-poly-
(ethyleneglycol) with the composition EO20PO70EO20 (pluronic
P123; M
w
¼ 5800, Sigma-Aldrich, St Louis, MO, USA) was used as
a surfactant template. For use as a swelling agent, 1,3,5-trime-
thylbenzene (mesitylene) was purchased from Wako Pure
Chemical Industries Ltd.
Preparation of recombinant glutaminase
In this study, N-terminal hexa-histidine-tagged g-glutamyl-
transpeptidase (i.e., glutaminase) from Pseudomonas nitro-
reducens IFO12694 was overexpressed using BL21 Star
Escherichia coli cells; subsequently, the puried enzyme was
used for encapsulation in mesoporous silicas and the microow
reaction experiment.
Genes of enzyme, vector, and bacterial strain
A complete glutaminase gene derived from Pseudomonas nitro-
reducens IFO12694, which consists of 1671 bp and encodes an
25
enzyme of 557 amino acids, was obtained as a synthetic gene
from Life Technologies Corp. (Carlsbad, CA, USA). The vector
pET302NT-His (Life Technologies Corp.) was used to clone the
glutaminase gene. E. coli. strain BL21 Star (DE3) (Life Tech-
nologies Corp.) was used as the bacterial strain for expression of
enzyme.
The entire coding sequence of glutaminase was cloned into
the vector pET302NT-His for expression in E. coli. The plasmid
was introduced into E. coli. strain BL21 Star (DE3), and the
ꢀ
bacterial cell was grown from a single colony overnight at 37 C
ꢁ
1
in 50 mL of LB broth (Sigma-Aldrich) containing 0.1 mg mL
ampicillin. Aer transferring to 2000 mL of Turbo Broth
Preparation of mesoporous silicas
As the mesoporous silica capsules for the glutaminase, we (Athena Enzyme Systems, Baltimore, MD, USA), containing
ꢁ1
ꢀ
prepared 2 types of SBA-15: SBA5.4 and SBA23.6, with pore 0.1 mg mL ampicillin, and growth for 3 h at 30 C, the cells
diameters of 5.4 and 23.6 nm, respectively.
were cultured with isopropyl b-D-thiogalactoside at a nal
26
ꢀ
SBA5.4 was synthesized from TEOS by using Pluronic P123,
concentration of 1 mM for 4 h at 30 C. The cells were harvested
ꢀ
as follows. In a typical synthesis, 10 g of the Pluronic P123 was by centrifugation at 7810 ꢂ g for 5 min at 4 C, and the pellet
dissolved in 300 mL of water, and the resultant mixture was was washed with 400 mL of cell wash buffer (20 mM Tris (2-
ꢀ
stirred overnight at 35 C. Then, 21.9 g of 12 N HCl and 21.3 g of amino-2-hydroxymethyl-1,3-propanediol
2 2 3
[H NC(CH OH) ])–
TEOS were added to the above solution. This mixture was stir- HCl buffer [pH 7.5] and 500 mM NaCl), and resuspended in
ꢀ
red for 20 h at 35 C. Aer stirring, the mixture was aged for 24 h 400 mL of cell lysis buffer (10 mM imidazole, 20 mM Tris–HCl
ꢀ
ꢁ1
at 35 C.
buffer [pH 7.5], containing 0.05 mg mL lysozyme, 1 mM
SBA23.6 was prepared according to the method reported by phenylmethylsulfonyl uoride, 500 mM NaCl, and 10 mM b-
27
Wang et al.,
with some modications. SBA23.6 was mercaptoethanol), and sonicated. The lysate was centrifuged at
9022 | RSC Adv., 2014, 4, 9021–9030
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ꢀ
7
810 ꢂ g for 20 min at 4 C. The supernatant containing over-
expressed enzyme was ltrated by a lter membrane with a 0.45
mm pore size and loaded into 2 mL Ni sepharose 6 fast ow resin
(GE Healthcare UK Ltd., Buckinghamshire, England) packed in
a chromatography column. Aer loading the sample in 8
columns, the expressed enzyme was washed with wash buffer
(
1
(
20 mM imidazole, 20 mM Tris–HCl [pH 7.5], 500 mM NaCl, and
0 mM b-mercaptoethanol), and eluted with elution buffer
500 mM imidazole, 20 mM Tris–HCl [pH 7.5], 500 mM NaCl,
and 10 mM b-mercaptoethanol). Subsequently, the sample
was dialyzed with dialysis buffer (50 mM Tris–HCl [pH 7.5],
300 mM NaCl, 1 mM EDTA, 15% glycerol, 0.05% NP-40, and 5 mM
b-mercaptoethanol), and inspissated by concentrating column.
The puried enzyme concentration was determined by the
bicinchoninic acid (BCA) method, with bovine serum albumin
as a standard; the purity of the enzyme was then evaluated by
SDS-polyacrylamide gel electrophoresis.
Adsorption of glutaminase to SBA mesopores
Fig. 1 Schematic illustration of the flow-type microreactor containing
A batch adsorption experiment was performed by combining 5 immobilised glutaminase–mesoporous silica (SBA) composite parti-
cles: (a) flow channel geometry of microreactor (top view) and (b) side
view of the microreactor. See also Fig. S1 (ESI†) for a detailed build
process of the microreactor and a characterisation of the microflow
channel.
mg of the mesoporous silica powder (SBA5.4 or SBA23.6) with
0.5 mL of 100 mM ethylamine hydrochloride solution (pH 5.5)
containing an appropriate amount of glutaminase (0.05–2.7 mg).
The glutaminase–SBA mixtures suspended in the solution were
ꢀ
gently shaken using a rotator for 17 h at 4 C. The glutaminase–
SBA composites were then centrifuged for 5 min at 15 300 ꢂ g
and the supernatant was recovered. In order to establish the
amount of glutaminase adsorbed to the SBAs, the enzyme
concentration of the rst supernatant was determined spectro-
photometrically by the BCA method, aer which the amount of
glutaminase adsorbed on the SBAs was determined by subtract-
ing the concentration of the glutaminase remaining in the
supernatant from that of the glutaminase before the
immobilisation.
Ltd., Tokyo, Japan) as adhesive and then sprinkled with
calcined SBA powder. It was then heated for 3 h at 85 C. The
SBA particles were then immobilised onto the surface by poly-
merization of a PDMS prepolymer layer (thickness: 0.1 mm); as
a result, the nal depth of the ow channel was about 0.1 mm
ꢀ
(inner volume: ca. 130 mL). Likewise, the SBA particles were
immobilised on the ow channel (width: 5 mm, length:
2
5
50 mm, depth: 0.1 mm) ditched in a PDMS membrane (width:
0 mm, length: 75 mm, thickness: 1 mm) by polymerization of a
For the evaluation of enzymatic activity in the glutaminase–
SBA composites in the batch experiment, the composite was
rinsed twice with the above ethylamine hydrochloride solution
and then resuspended in an appropriate volume of the solution
so that the nal concentration of the immobilised enzyme was
PDMS prepolymer layer (thickness: 0.1 mm), resulting in a
depthless ow channel. The SBA particle-immobilised surfaces
of the glass channel and PDMS membrane were air-dried to
remove any unattached SBA, aer which the lower ow channel
was sealed with the upper PDMS membrane. The ow channel
was tted with 2 microtubes for supplying solution.
ꢁ
1
0
.5 mg mL .
Recombinant glutaminase was encapsulated in the pores of
the SBA-15 immobilised in the microreactor by adding 1 mL of
Preparation of a microreactor containing glutaminase–SBA
composites
1
00 mM ethylamine hydrochloride solution (pH 5.5) containing
ꢁ
1
ꢁ1
The proposed design of the polydimethylsiloxane (PDMS)/glass 0.2 mg mL glutaminase into the ow channel at 10 mL min
ꢀ
microreactor containing the glutaminase–mesoporous silica at 4 C using a syringe pump (Model 11 plus; Harvard Appa-
(
SBA-15) composite particles is shown schematically in Fig. 1. ratus, Holliston, MA, USA). The obtained glutaminase–SBA
The SBA particles were immobilised over the entire surface of composites immobilised in the reactor were then rinsed with
ꢁ
1
both sides of the slit in the microreactor, that is, the top and 1 mL of above ethylamine hydrochloride solution at 20 mL min
bottom of the ow channel, as follows. A glass substrate (width:
.
5
2 mm, length: 76 mm, thickness: 1 mm; Matsunami Glass
Visualization of glutaminase–SBA composites
Ind., Ltd, Osaka, Japan) was used as the base material to
fabricate the ow channel of the microreactor. To immobilise To allow determination of the amount of glutaminase
SBA particles, the glass surface of the ow channel (Fig. 1a; adsorbed onto the SBA particles, the enzyme was chemically
width: 5 mm, length: 250 mm, depth: 0.2 mm), fabricated by modied with a thiol-reactive uorescent dye (Alexa Fluor 488
max max
etching the glass substrate, was coated with a thin layer of
C
5
-maleimide, C30
H
25
N
4
NaO12
S
2
; M
r
720.66; labs /lem
¼
liquid PDMS prepolymer (SILPOT 184; Dow Corning Toray Co., 493 nm/516 nm; Life Technologies Corp.) at the thiol group
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position on the enzyme, with a degree of labelling of
The enzymatic activities of glutaminase–SBA (SBA5.4 or
1
3
approximately 1 dye molecule to 1 enzyme molecule. Aer SBA23.6) composites in the batch experiment were measured
preparing the uorescently labelled enzyme, the enzyme was for the synthesis reaction forming L-theanine from 2 substrates,
immobilised in the SBA-xed microreactor by the above- 20 mM glutamine and 100 mM ethylamine (pH 4, 6, 8, 10, and
mentioned encapsulating and washing procedure. Then, the 12). A 1.1 mL sample of the reaction mixture containing 50 mg
amount of glutaminase immobilised in each microreactor glutaminase in the composite form, along with the substrates,
ꢀ
was estimated from standard curves of uorescence intensi- was gently shaken using a rotator for 1 h at 20 C. The reaction
ties of Alexa 488 dye in the dye-labelled glutaminase–SBA was terminated by adding perchloric acid, aer which gluta-
composites (0.003–3 mg glutaminase) using a uorescence- minase activity was evaluated by determining the amounts of
image analyser (FLA-5100; FUJIFILM Corp., Tokyo, Japan).
L-theanine and L-glutamic acid as a by-product using a high-
Evaluation of the encapsulation of the enzyme in SBA performance liquid chromatograph (LaChrom Elite; Hitachi
particles immobilised in the microreactor was also carried out High-Technologies Corp.) equipped with an octadecylsilyl-
by direct visualization using a uorescence microscopy tech- column (LaChrom C18-AQ; particle diameter: 5 mm, Hitachi
nique. The existence and relative localization of the enzyme High-Technologies Corp.). The sample component was sepa-
encapsulated in the pores could be monitored by detecting the rated with the mobile phase (liquid composition; H
2
O–meth-
26,30
ꢁ1

uorescently labelled version of the enzyme.
The gluta- anol–triuoroacetic acid ¼ 980 : 20 : 1) at 0.5 mL min for
ꢀ
minase–SBA composites immobilised in the microreactor were 12 min at 30 C, and the detection of L-theanine and L-glutamic
observed by a combination of differential interference contrast acid was performed using a UV detector at 210 nm. For
(DIC) and uorescence microscopy using an inverted micro- comparison purposes, the enzymatic activities of the free
scope (ECLIPSE TE2000-U; Nikon Corp., Tokyo, Japan) equip- enzymes without mesoporous silica were measured. The enzy-
ped with a 100ꢂ, 1.49 numerical aperture (NA) oil-immersion matic activity was characterised in terms of conversion ratio (CR)
objective lens. The excitation and emission wavelengths were which is calculated using the following equation (eqn (1)):
selected with the lter set GFP-BP (suitable for Alexa 488, EX480/
cðtheanineÞ
40, DM505, and BA535/50) produced by Nikon. A uorescence
CRð%Þ ¼ c
ꢂ 100
(1)
0
ðglutamineÞ
0
(glutamine) is the initial glutamine concentration
image of the labelled glutaminase was captured on an electron-
multiplying charge-coupled device (EM-CCD) camera (ImagEM; where c
C-9100-13, Hamamatsu Photonics K.K., Shizuoka, Japan) and (20 mM), c(theanine) is the theanine concentration aer the
recorded using an image processor (AQUACOSMOS analysis g-glutamyl transfer reaction.
soware; Hamamatsu Photonics K.K.). Simultaneously, the
In order to assess the ow system, the glutamine–ethylamine
corresponding image of mesoporous silica particles, used as reaction solution was added to the ow channel containing the
capsules for the enzyme, was independently obtained by DIC immobilised enzyme. The substrate solution (20 mM gluta-
microscopy.
mine–100 mM ethylamine) was passed through the reactor at 5
or 10 mL min , and the theanine synthesis by the immobilised
ꢁ
1
glutaminase was carried out at various reaction temperatures
Enzymatic reactions in batch and microreactor
ꢀ
(4, 10, 20, 30, 40, and 50 C) and pH of substrate solutions (pH 4
Theanine synthesis by glutaminase was used as a model reac-
tion in batch and microow reactions. Theanine, which has
been utilised as a food additive and dietary supplement, can be
synthesized efficiently by a g-glutamyl transfer reaction using
and 10). Recovery of product solution from the outlet of the ow
channel commenced aer an initial 300 mL pre-delivery delay of
substrate solution using a syringe pump to reach the steady-
state ow in the microreactor. In cases where the entire
microreactor system, including substrate supply, the main unit
of the reactor, and the product collection area, were heated
25,31
glutaminase under basic reaction conditions (pH 10–11).
Because a mixture of 2 substrates acting as a g-glutamyl donor–
acceptor pair is required, in this work, a combination of gluta-
mine (donor) and ethylamine (acceptor) was used. The reaction
proceeds via g-glutamyl transfer from glutamine to ethylamine,
with a suppression of the hydrolysis of glutamine, i.e., conver-
(total heating), the temperature in the system was controlled
using an air heat-type incubator (MIR-154; SANYO Electric Co.,
Ltd., Osaka, Japan). In cases where the main unit of the reactor
within the microreactor system was locally heated (local heat-
ing), the temperature in the main reactor was controlled by
temperature control units (Stage top incubator, INU-ONICS;
Tokai Hit Co., Ltd., Shizuoka, Japan), while both the substrate
32,33
sion of L-glutamine to L-theanine (Scheme 1).
ꢀ
supply and the product collection area were kept at 4 C, using
the air incubator mentioned above (Fig. S2, ESI†). Temperatures
ꢀ
ꢀ
were made to alternate between 4 C and 30 C during the
continuous microow reaction by powering the temperature
control units equipped with the microreactor off or on,
respectively. The pH values were made to alternate between
pH 4 and 10 by changing the pH of the delivery solution. Aer
the initial 300 mL pre-delivery, the substrate solutions were
passed through the microreactor for 20 straight assays without
Scheme 1 Reaction scheme of L-theanine synthesis by glutaminase.
The theanine is obtained by g-glutamyl transfer reaction from gluta-
mine to ethylamine with glutaminase.
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a
pre-delivery solution again. Aer the reaction, the produced Table 1 Structural properties of SBA-15-type mesoporous silicas
L-theanine solutions were collected from the outlet of the
microreactor and the glutaminase activity was evaluated by
the measuring method previously described.
Specic
Total pore
volume
Types of Pore
SBA-15
surface 2
Particle
morphologies
ꢁ1
3
ꢁ1
diameter (nm) area (m g
)
(cm g
)
SBA5.4
SBA23.6 23.6
5.4
643.5
470.0
0.61
2.17
Rod
Sphere
Results and discussion
Effect of pore size of mesoporous silicas on adsorption of
a
See also Fig. 2 for nitrogen adsorption–desorption isotherms and the
corresponding pore size distribution curves for SBA5.4 and SBA23.6.
enzyme and the activity in batch reaction
As the mesoporous silica capsules for the enzyme, we prepared
two SBA-15-type mesoporous silicas (SBA5.4 and SBA23.6). The
ordered mesopores of calcined SBAs were conrmed by
measuring nitrogen adsorption–desorption isotherms and the
corresponding pore-size distribution curves of dried powder
samples (Fig. 2). The structural parameters and the SEM images
of the SBA series are shown in Table 1 and Fig. 3, respectively. As
shown in Table 1 and Fig. 2b, the pore diameters of SBA5.4 and
SBA23.6 were 5.4 nm and 23.6 nm, respectively. These data
indicated that SBAs with different pore sizes were successfully
prepared, although the particle morphologies of SBAs were
considerably different, as is clear from the SEM images of
SBA5.4 (Fig. 3a, rod-like) and SBA23.6 (Fig. 3b, sphere with 4 mm
particle diameter).
Fig. 3 Scanning electron microscope images of SBA-15-type meso-
porous silicas: (a) SBA5.4 and (b) SBA23.6. The scale bar is 10 mm.
Before assessing theanine synthesis in the glutaminase–SBA
composite contained in microreactor, batch experiments were
carried out to investigate the enzyme-encapsulation efficacy and
catalytic function under high pH conditions (Fig. 4). In this
study, the engineered glutaminase was prepared as an enzyme
solution of high purity and used for the experiments.
Fig. 4 (a) Adsorption isotherms of glutaminase on SBAs. The amounts
of glutaminase adsorbed to SBA5.4 (white squares) and SBA23.6 (black
squares) in 100 mM ethylamine hydrochloride solution (pH 5.5)
were measured spectrophotometrically with respect to the equilib-
rium concentrations of glutaminase. (b) Conversion of L-glutamine to
L-theanine in batch reactions by free glutaminase (slash bar), gluta-
minase–SBA5.4 composite (white bar), and glutaminase–SBA23.6
composite (black bar). Reaction conditions: enzyme content, 50 mg;
composition of substrate, 20 mM glutamine–100 mM ethylamine
Fig. 2 (a) Nitrogen adsorption–desorption isotherms and (b) corre- (pH 10); total volume of reaction mixture, 1.1 mL; reaction time, 1 h;
ꢀ
sponding pore size (dp) distribution curves obtained from the reaction temperature, 20 C. See also Fig. S3 (ESI†) for effect of
adsorption branch by the Barret-Joyner-Halenda (BJH) method for reaction temperature on conversion to L-theanine in batch reactions
SBA5.4 (white squares) and SBA23.6 (black squares).
by free glutaminase and glutaminase–SBA23.6 composite.
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Paper
12,16
In encapsulation of glutaminase in the mesopores of SBAs, encapsulated in FSM and HOM-type mesoporous silicas.

rst, the effect of pore sizes of the 2 SBAs on the amounts of The efficiency of the glutaminase–SBA23.6 composite in
glutaminase adsorbed on the SBA pores in 100 mM ethylamine synthesis of L-theanine at various reaction temperatures (a
ꢀ
hydrochloride solution (pH 5.5) was conrmed by a batch temperature range of 4–50 C) was also of a comparable level to
experiment (Fig. 4a). As shown in Fig. 4a, the amount of that of the free enzyme (Fig. S3, ESI†), indicating that the
glutaminase adsorbed to SBA23.6 was notably larger than that mesoporous framework of SBA23.6 did not inhibit the theanine
adsorbed to SBA5.4, indicating that the amount of adsorbed synthesis by glutaminase regardless of the reaction
enzyme was greatly dependent on the pore size of the SBAs. The temperature.
proportion of glutaminase adsorbed onto the pores of the
From these results, we considered that the pore size of
SBA23.6 was found to be up to approximately 10 wt% (by weight SBA23.6 was more efficient for encapsulating glutaminase and
of SBA23.6), which was 3 times higher than that of the gluta- conducting theanine synthesis. Thus, the SBA-15 microsphere
minase adsorbed to SBA5.4. From this result, it could be (i.e., SBA23.6) was used for the subsequent enzyme encapsula-
surmised that it is difficult to encapsulate the enzyme easily in tion and the microow reaction using the enzyme microreactor.
the pores of SBA5.4 with a 5.4 nm pore diameter, because
34
glutaminase is approximately 5–6 nm in dimension; that is,
pores larger than the size of the enzyme is required for efficient
encapsulation of enzyme. However, the maximum amount of
glutaminase adsorbed on SBA23.6 (10 wt.%) (Fig. 4a) was lower
than that adsorbed on TMPS10.6 (Taiyo Kagaku Meso Porous
Silica, 10.6 nm pore diameter) (14 wt%), which was used in our
previous study. However, the TMPS10.6 was modied with a
zirconia compound and then treated with 3-glycidoxypropyl-
trimethoxysilane to efficiently immobilise more enzyme.
Although the amount of immobilised enzyme in the unmodi-
Evaluation of conversion and selectivity in theanine synthesis
by glutaminase–SBA composites
To examine the optimal substrate solution for synthesis of L-the-
anine by the encapsulated enzyme, the effect of pH value of the
substrate solution containing 20 mM glutamine and 100 mM
ethylamineontheconversionsofL-glutaminetoL-theaninebyfree
glutaminase and glutaminase–SBA23.6 composite was
conrmed in batch experiments (Table 2). As shown in Table 2,
theanine production was predominant at pH 10 in the cases of
both free glutaminase and glutaminase–SBA23.6 composite. The
optimal pH value for simultaneous hydrolysis of glutamine, i.e.,
conversion of L-glutamine to L-glutamic acid, was pH 8. Interest-
ingly, conversions to L-glutamic acid as a by-product by the free
glutaminase and the glutaminase–SBA23.6 composite at pH 10
were 37.7 and 2.5%, respectively. Therefore, it was found that the
selectivity of L-theanine in the glutaminase–SBA23.6 composite
13

ed SBA23.6 was approximately 70% of that in modied
TMPS10.6, the enzyme did not leach from the enzyme–SBA23.6
composite during the 2 wash steps despite the non-chemical
surface modication of the pores. This indicates that the mes-
opores of the calcined SBA do not require pre-treatment (such
as chemical cross-linking between the enzyme and solid
support) to prevent enzyme leaching. Therefore, SBA23.6 is a
relatively cost-efficient support material for reagent-free enzyme
immobilisation and is superior to modied TMPS10.6.
The enzymatic activities of the obtained glutaminase–SBA
composites were next measured in batch reactions (Fig. 4b)
before attempting an actual microow reaction with a micro-
reactor containing the composites. The batch enzymatic reac-
tions using the glutaminase–SBA composites had to be carried
out at a moderate mixing speed, because the mesoporous
framework degrades under vigorous stirring because of its low
mechanical strength. In the batch experiments, the enzymes
that were adsorbed to mesopores of SBAs demonstrated
different activities. As shown in Fig. 4b, the glutaminase–SBA5.4
composite had 47.8% of the activity of the native SBA-free
glutaminase in theanine production. On the other hand, the
glutaminase–SBA23.6 composite had activity comparable to
that of the free glutaminase (retained activity: 91.3% of that for
the free enzyme), even aer the adsorption. These results
(94.4%) was markedly higher than that of free glutaminase
without SBA (54.9%) under these reaction conditions. This
difference in enzymatic selectivity behaviour with and without
mesoporous silica may be attributed to the difference in accessi-
bility of water molecules to the active centre of the enzymes
encapsulated in the mesopores; that is, it can be presumed that
water molecules, which are required for glutamine hydrolysis,
could not diffuse extensively into the pores of SBA, because of the
high hydrophobicity of SBA-15-type mesoporous silica (hydro-
phobicity for pore surface of SBA > that of FSM). Thus, we
demonstrated the selective conversion to L-theanine by gluta-
minase–SBA23.6 composite as far as the theanine synthesis
reaction between the glutamine, the ethylamine and the gluta-
minase derived from Pseudomonas nitroreducens is concerned.
35
Direct observation of glutaminase immobilised in
microreactor
indicated that the difference of the pore size between SBA5.4 In order to directly conrm the specic and stable encapsula-
and SBA23.6 had a considerable effect on the activity of gluta- tion of glutaminase by the SBA23.6 in the ow-type micro-
minase under these reaction conditions. The higher remaining reactor, uorescently labelled enzyme adsorbed to SBA
activity for the glutaminase–SBA23.6 composite was likely due microspheres in the reactor was observed by uorescence
to the larger pores of SBA23.6 and the structure of the meso- microscopy containing DIC with a high-sensitivity EM-CCD
porous channels. In other words, this suggests that the strength camera. Fig. 5 shows a picture of the enzyme microreactor
of substrate-immobilised enzyme interactions can be enhanced fabricated in this study (Fig. 5a) and the corresponding uo-
by the efficient introduction of the substrate into the SBAs with rescence images of glutaminase–SBA23.6 composite particles
larger pores. Similar results have been reported for lipase immobilised in the microreactor (Fig. 5b and c). First, the
9026 | RSC Adv., 2014, 4, 9021–9030
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Table 2 Effect of pH value of substrate solution on conversion ratio and selectivity in theanine production in batch reactions by free glutaminase
a
and glutaminase–SBA23.6 composite
Free glutaminase
Conversion
Glutaminase–SBA23.6
Selectivity
Conversion
Selectivity
pH of react.
soln
Theanine (%)
Glutamic acid (%)
Theanine (%)
Theanine (%)
Glutamic acid (%)
Theanine (%)
4
6
8
1
1
0
11.8
70.6
78.8
37.7
3.0
0
0
0
0.8
41.8
0
0
—
0
4.7
94.4
—
0.3
0.8
45.8
0
0.4
1.0
54.9
0
9.0
16.2
2.5
0
0
2
a
Reaction conditions: enzyme content, 50 mg; composition of substrate, 20 mM glutamine–100 mM ethylamine (pH 4, 6, 8, 10, and 12); total volume
ꢀ
of reaction mixture, 1.1 mL; reaction time, 1 h; reaction temperature, 20 C. The conversions to L-theanine by free glutaminase and glutaminase–
SBA23.6 composite at pH 10 correspond to those shown in Fig. 4b.
immobilised in the ow channel by direct visualization using
optical microscopy. The xed SBA23.6 particles with a 4 mm
particle diameter in 100 mM ethylamine hydrochloride solution
(pH 5.5) were successfully observed individually by DIC
microscopy (Fig. 5c, upper). Next, green emission due to the
Alexa 488 dye was conrmed in a same microscope eld of the
SBA particles when they were irradiated with an excitation light
(480/40 nm; Fig. 5c, lower panel), indicating that glutaminase
molecules were present in the particles. As observed from the
microscopic image of the Alexa 488 dye-labelled glutaminase,
the area of uorescence emission from the enzyme was
distributed evenly across the microspheres, suggesting that the
distribution of glutaminase in the silica particle was uniform.
This uniform distribution was attributed to the successful
encapsulation of the enzyme in the SBA23.6 mesopores. As
reported previously, if the labelled enzymes were adsorbed
mainly on the surfaces of the mesoporous silica particles with
small pore sizes, uorescent enhancement would be obtained
Fig.
5 Direct visualization of glutaminase–SBA23.6 composites
immobilised in microreactor: (a) picture of the microreactor, (b) the
corresponding fluorescence image of Alexa 488 dye-labelled gluta-
minase in the microflow channel as captured by fluorescence-image
analyser, (c) differential interference contrast (DIC) image of SBA23.6 from the edges of the particles, because enzymes with sizes
particles immobilised in the flow channel (upper) and the corre-
larger than the pores of the silica particles cannot penetrate
beyond the entrance of the pores. In Fig. 5c (lower panel), a
sponding fluorescence image of Alexa 488 dye-labelled glutaminase
encapsulated in SBA23.6 (lower) observed by optical microscopy at the
position indicated by a red arrow of (b), and (d) schematic illustration of
enzyme–mesoporous material microsphere composite. The scale bar
is 10 mm.
36
high signal-to-noise ratio for the image of the labelled enzymes
was obtained and maintained during real-time observation,
resulting from both the specic adsorption of the labelled
enzyme to the silica particle rather than to the PDMS membrane
in the ow channel and suppression of the leaching of the
enzyme from the immobilised composite into the solution.
From the abovementioned experiments, based on the direct
visualization of glutaminase–SBA23.6 composites, we presumed
that the pore size of the SBA23.6 is suitable for encapsulating
the enzyme and that the enzymes are indeed encapsulated in
the SBA microsphere, as shown in Fig. 5d.
pattern of immobilisation area for the Alexa Fluor 488 dye-
labelled glutaminase in the microreactor was conrmed by
scanning the entire microreactor with a uorescence-image
analyser (Fig. 5b). The area of uorescence emission from the
enzyme was distributed evenly from inlet to outlet in the
microow channel, suggesting that the distribution of the
glutaminase–SBA23.6 composites in the channel was uniform.
The amount of the immobilised glutaminase per unit area was
ꢁ
2
Evaluation of theanine synthesis in enzyme microreactor
estimated to be approximately 20 ng mm from the standard
curve of uorescence intensities of Alexa 488 dye-labelled We next attempted to use a ow-type microreactor containing
glutaminase–SBA23.6 composites determined by the above the glutaminase–SBA23.6 composites for continuous synthesis
image analyser.
of L-theanine. In the industrial process of L-theanine production
We further evaluated the encapsulation and the relative with ethylamine as a g-glutamyl acceptor, it is necessary to carry
localization of the glutaminase in SBA23.6 particles out the synthesis reaction at a low temperature owing to the low
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ꢀ
boiling point of ethylamine (16.6 C). Therefore, the entire theanine (Fig. 6a). As is clear from the gure, interestingly,
microreactor system, including the substrate supply (inlet), higher conversion ratios were obtained at each temperature,
ꢀ
main unit of reactor, and product collection area (outlet), were except for 4 C, when the main unit of the reactor was heated
ꢀ
chilled to 4 C during the theanine synthesis. Further, in order with local temperature control units, compared to use of total
ꢀ
to enhance the enzymatic activity, local control of the reaction heating. At the optimal reaction temperature (30 C) for the-
ꢀ
temperature (4–50 C) in the enzyme-encapsulated microow anine synthesis, the conversion ratio in the case of local heating
channel was attempted (Fig. 1b).
was approximately 2 times higher than that in the case of total
In the ow system in this study, rst, a reaction solution heating. From these results, it can be speculated that the higher
containing the 2 substrates (20 mM glutamine–100 mM ethyl- activity shown in local heating of the glutaminase–SBA23.6
amine, pH 10) was added into the ow channel containing the composites in the microreactor is associated with suppression
ꢁ1
immobilised enzyme (ow rate of substrate: 10 mL min ). We of the production of gas-phase ethylamine in the substrate
ꢀ
examined whether various reaction temperatures (4, 10, 20, 30, supply unit, given the low boiling point of ethylamine (16.6 C),
ꢀ
4
0, and 50 C) with total or local heating of the enzymatic during the theanine synthesis reaction. From Fig. 6a, it was
reaction eld in the microreactor affected the conversion to L- found that the activities of the immobilised enzymes increased
ꢀ
ꢀ
ꢀ
by local heating in the following order: 30 C > 20 C > 10 C > 40
ꢀ
ꢀ
ꢀ
C > 4 C > 50 C. On the other hand, the activity measured when
the entire microreactor system, including the substrate supply,
main unit of reactor, and product collection area, was heated to
ꢀ
ꢀ
4
0 C, was lower than that at 4 C, and no production of the-
ꢀ
anine was obtained at 50 C. Furthermore, the conversion ratio
at 40 C in the case of local heating was approximately 4 times
ꢀ
higher than that in the case of total heating, supporting the
above hypothesis that the ethylamine is vaporised at a
ꢀ
temperature range of 10–50 C.
To investigate precise regulation of L-theanine synthesis in
the continuous microow reaction, in addition to the above
advantage of local temperature control in the reaction system,
we also examined the inuence of changes of both reaction
temperature and pH value of the substrate solution, containing
20 mM glutamine and 100 mM ethylamine, on the conversions
of L-glutamine to L-theanine in glutaminase–SBA23.6
a
composite-xed microreactor (Fig. 6b). When the enzymatic
activity of the immobilised glutaminase at staggered tempera-
ꢀ
tures (i.e., 4 and 30 C) during the continuous theanine
synthesis at pH 10 was evaluated with local heating (ow rate of
ꢁ
1
substrate: 5 mL min ), the glutaminase–SBA23.6 composites
contained in the microreactor indicated alternately the gradual
increase and decrease of the conversion to L-theanine at 30 and
ꢀ
4
C, respectively. The maximum conversion ratio in this
ꢀ
ꢁ1
microreactor (at 30 C and a ow rate of 5 mL min ) was
approximately 50%, which was higher than that of the prototype
enzymatic microreactor with surface-modied TMPS10.6 used
13
in our previous study (maximum conversion ratio: ca. 10%).
Fig. 6 Analyses of L-theanine synthesized using a microreactor con-
taining glutaminase–SBA23.6 composites and its temperature and pH
control: (a) effect of reaction temperature with total or local heating of
enzymatic reaction field in the microreactor on the conversion of L-
When the effect of conversion to L-theanine on residence time
was examined, an increase in conversion in proportion to the
residence times was conrmed. A similar tendency was
glutamine to L-theanine. Reaction conditions: enzyme content, ca. 50 observed with both the glutaminase–SBA23.6 composite-xed
mg; composition of substrate, 20 mM glutamine–100 mM ethylamine
microreactor and the glutaminase–TMPS10.6 composite-xed
ꢁ
1
(
pH 10); flow rate of substrate solution: 10 mL min ; reaction time, 30
microreactor, indicating that the performance of the current
enzymatic microreactor was equivalent to that of the prototype
microreactor despite the non-chemical surface modication of
min. The conversions to L-theanine were measured thrice per exper-
imental point (N ¼ 3). (b) Effect of reaction temperature and pH of
substrate solution on the theanine production during the continuous
reaction by local heating of enzymatic reaction field in the micro- the pores (Fig. S4, ESI†). This increased conversion in the new
reactor. Reaction conditions: composition of substrate, 20 mM
glutamine–100 mM ethylamine (pH 10 constant in the case of
enzyme microreactor was due to both the improved micro-
channel length and the optimised pore size of the silica support
temperature control, pH 4 or 10 in the case of pH control); reaction
ꢀ
ꢀ
for reagent-free, stable enzyme immobilisation. Moreover, we
could terminate the theanine production completely by rapid
temperature, 4 or 30 C in the case of temperature control, 30
C
constant in the case of pH control; flow rate of substrate solution: 5 mL
ꢁ1
ꢀ
min ; reaction time, 15 min.
cooling to the vicinity of 0 C, resulting in temporary off-
9028 | RSC Adv., 2014, 4, 9021–9030
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regulation of the enzymatic activity. In addition, as was the case glutaminase–SBA23.6 microreactor allowed a marked increase
with temperature control, alternately supplied substrate solu- in conversion ratio and precise regulation of L-theanine
tion with a different value of pH (i.e., pH 4 and 10) led to uc- synthesis during continuous theanine production. Control of
tuation of the conversion. The behaviour indicated in the above enzyme activity was achieved by means of local heating of the
results suggested that the efficiency of the enzymatic reaction reaction eld in the ow channel, so avoiding effects on
could be controlled accurately by adjusting both the reaction temperature-sensitive reactants, and by controlling the pH
temperature and the pH value of the substrate solution during value of the substrate solution during solution supply. Hence,
the microow reactions. However, the conversion ratios at the this novel microuidic reactor system using mesoporous silica
same temperature and pH range were not equivalent. This as a scaffold for enzyme-immobilisation enables efficient
suggests that, under these experimental conditions, it was continuous synthesis of functional compounds, even from
difficult to instantaneously maximize enzymatic activity by reactive substrates with low boiling points and low thermal
controlling the temperature and pH in the continuous micro- stability, and effective on-off regulation of enzymatic activity by

ow reaction because a specic incubation time or substrate means of accurate control of the reaction temperature and the
solution was required to reach the desired temperature and pH pH in the micro-reaction eld.
in areas surrounding the encapsulated enzyme. When
comparing between the 2 cases, a maximum conversion ratio in
the case of temperature control was clearly higher than that in
Acknowledgements
the case of pH control, even when maintaining the same reac-
ꢀ
tion conditions (in pH 10 at 30 C). We considered that the This research was nancially supported by the Adaptable and
lower enzymatic activity shown with the pH change is because Seamless Technology Transfer Program through Target-driven
the pH environment surrounding the active centre of the R&D (A-STEP, no. AS231Z03383E) of the Japan Science and
enzyme in the pores of mesoporous silica does not reach the Technology Agency (JST). The authors would like to thank Dr
optimal pH for theanine synthesis, owing to the particular space Takuji Yokoyama (Taiyo Kagaku) and Dr Takayuki Y. Nara
within the mesopores. Therefore, it can be said that the regu- (AIST) for providing helpful advice.
lation of reaction temperature in the microow reaction could
affect and control enzyme activity more quickly and easily than
regulation via reaction pH. The alternate on-off regulation of
enzymatic activity during prolonged addition of the substrate
Notes and references
solution in 20 assays observed in Fig. 6b may be due to retention
of a stable immobilised state of glutaminase; that is, it can be
suggested that enzyme molecules encapsulated in the pores do
not experience the reduced activity associated with the leaching
of the enzyme from the ow channel during continuous
reactions.
Thus, these results suggested that signicant enhance-
ment of the enzymatic activity in the enzyme–mesoporous
silica composite provides the possibility of high productivity
of L-theanine, by using this novel enzyme microreactor, and
due to the sustained operational stability of the composites
immobilised in the ow channel, which also facilitated
retention of the activity of the encapsulated enzyme.
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