Improvement of aldehyde tolerance and sequential aldol condensation activity of deoxyriboaldolase via immobilization on interparticle pore type mesoporous silica

Article abstract of DOI:10.1016/j.molcatb.2010.10.008

Deoxyriboaldolase from Klebsiella pneumonia (KpDERA) was immobilized on interparticle pore type mesoporous silica (IMS), which consists of aggregates of hexagonally close-packed silica nanoparticles. Incubation of the free KpDERA in 300 mM acetaldehyde resulted in a decrease in the free enzyme activity to 20% of the initial activity after incubation for 30 min. No activity was observed after 90 min incubation. In contrast, the IMS-immobilized KpDERA retained more than 50% of its activity after 30 min and 11% activity after 90 min incubation. The product yield of a sequential aldol condensation reaction, which is central to the production of pharmaceutically important intermediates, was found to be higher for IMS-immobilized KpDERA than for the free enzyme. These results suggest the potential utility of IMS as an immobilization support for DERA.

Full text of DOI:10.1016/j.molcatb.2010.10.008

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 181–186
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Improvement of aldehyde tolerance and sequential aldol condensation activity of
deoxyriboaldolase via immobilization on interparticle pore type mesoporous
silica
a
a,1
a
b
a
a
Takayuki Y. Nara , Hideaki Togashi , Seigo Ono , Miki Egami , Chisato Sekikawa , Yo-hei Suzuki ,
a
c
c
c
a
a,∗
Isao Masuda , Jun Ogawa , Nobuyuki Horinouchi , Sakayu Shimizu , Fujio Mizukami , Tatsuo Tsunoda
a
Research Center for Compact Chemical Process, AIST, Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan
JGC Catalysis and Chemicals Ltd., 13-2 Kitaminato, Wakamatsu, Kitakyushu 808-0027, Japan
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2010
Received in revised form 26 October 2010
Accepted 30 October 2010
Available online 9 November 2010
Deoxyriboaldolase from Klebsiella pneumonia (KpDERA) was immobilized on interparticle pore type
mesoporous silica (IMS), which consists of aggregates of hexagonally close-packed silica nanoparticles.
Incubation of the free KpDERA in 300 mM acetaldehyde resulted in a decrease in the free enzyme activity
to 20% of the initial activity after incubation for 30 min. No activity was observed after 90 min incubation.
In contrast, the IMS-immobilized KpDERA retained more than 50% of its activity after 30 min and 11%
activity after 90 min incubation. The product yield of a sequential aldol condensation reaction, which
is central to the production of pharmaceutically important intermediates, was found to be higher for
IMS-immobilized KpDERA than for the free enzyme. These results suggest the potential utility of IMS as
an immobilization support for DERA.
Keywords:
Deoxyriboaldolase
Interparticle type mesoporous silica
Immobilized enzyme
Aldehyde tolerance
Aldol condensation
© 2010 Elsevier B.V. All rights reserved.
1
. Introduction
An enzyme is a biocatalyst with several advantageous proper-
to a substituted acetaldehyde to form 3-hydroxy-4-substituted
butyraldehyde, followed by reaction with a third acetaldehyde.
The obtained 2,4,6-trideoxyhexoses are valuable chiral intermedi-
ates for the production of pharmaceutical products, for example
a cholesterol-lowering drug [7–10]. DERA-catalyzed reactions
may provide an alternative to chemical methodologies used for
the synthesis of these intermediates. However, DERA is labile at
high aldehyde concentrations, and a large quantity of enzyme is
required to obtain useful product yields [10]. Unless the toler-
ance for aldehydes is improved, DERA may not be practical for
commercial industrial applications.
ties, including extremely high catalytic activity, substrate-, stereo-,
regio-, and reaction-specificity, and a low environmental load [1,2].
Enzymes have emerged as important catalysts for the industrial
synthesis of bulk chemicals, pharmaceutical intermediates, food
ingredients, and agricultural chemicals [2–4].
Deoxyriboaldolase (DERA; EC 4.1.2.4) catalyzes a reversible
aldol reaction that generates 2-deoxy-d-ribose-5-phosphate
(
DR5P) from d-glyceraldehyde 3-phosphate (G3P) and acetalde-
hyde [5]. DERA is unique in catalyzing aldol condensations between
two or more aldehydes, and its broad substrate specificity confers
considerable utility as a biocatalyst [6]. Gijsen and Wong [7]
reported the double aldol condensation between three acetalde-
hyde molecules catalyzed by DERA from Escherichia coli (EcDERA).
The reaction began with the stereospecific addition of acetaldehyde
Enzyme immobilization on a solid support offers several advan-
tages over catalysis methods that employ free enzymes. Solid
supports can enhance the enzyme stability, facilitate separa-
tion, may be reused, and permit continuous operation. Many
immobilization techniques, such as physical adsorption, covalent
attachment, and encapsulation, as well as organic and inorganic
supports for enzyme immobilization have been studied [2,11–13].
No single technique or support has emerged as a universal stan-
dard, meaning that the technique and support must be optimized
for each application.
Abbreviations: DERA, deoxyriboaldolase; IMS, interparticle pore type meso-
porous silica; DR5P, 2-deoxyribose 5-phosphate.
∗ Corresponding author. Tel.: +81 29 861 4633; fax: +81 29 861 4633.
Interparticle pore type mesoporous silica (IMS) consists of
hexagonally close-packed silica nanoparticle aggregates. The
aggregated nanoparticles form micron-size particles. IMS parti-
E-mail address: t.tsunoda@aist.go.jp (T. Tsunoda).
Present address: JGC Corporation, 2205 Narita, Oarai, Ibaraki 311-1313, Japan.
1
1
381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.10.008

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cles contain mesopores with tetrahedral symmetry defined by the
interstitial spaces between nanoparticles. IMS particles possess
excellent features as a support for enzyme immobilization. The
pore size (2–50 nm) can be controlled via the size of the silica
nanoparticlesused, theparticles are inexpensive tosynthesize, they
are chemically inert, and they exhibit a high mechanical stabil-
ity. The surfaces of IMS particles display silanol groups to which
biomolecules may bind via electrostatic interactions. The surface
silanol groups can be modified using a variety of organosilanes
to alter the surface charge and/or form covalent bonds with the
immobilized biomolecules [14].
centration gradient of 10–500 mM imidazole in 20 mM Tris–HCl
(pH 7.5), 500 mM NaCl. The peak fractions were collected and
dialyzed against 50 mM TEA (pH 7.5). The purity of the enzyme
was verified by sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE).
2.3. Characterization
Nitrogen adsorption/desorption isotherms were measured at
the temperature of liquid N (77 K) using the AUTOSORB-1 analyzer
2
(Quantachrome Instruments, FL, USA). Prior to measurement, the
◦
In the present study, DERA from Klebsiella pneumonia (KpDERA)
samples were degassed at 200 C for 3 h under vacuum. The specific
[
15] was overexpressed and immobilized on IMS, and the appli-
surface area was calculated by the Brunaur–Emmett–Teller (BET)
method using adsorption data over the relative pressure range of
cability of IMS as an enzyme immobilization support was tested.
The IMS-immobilized KpDERA showed improved tolerance toward
high concentrationsof acetaldehydes. The aldol condensationprod-
uct yield was higher than that obtained using free enzymes.
P/P = 0.05–0.30. The total pore volume was estimated from the
0
quantity ofN adsorbedatthe maximum relativepressure. The pore
2
size distribution was determined by the Barrett–Joyner–Halenda
(BJH) method using the adsorption or desorption branches. To study
the changes in the nitrogen adsorption isotherms and pore size
distributions by KpDERA immobilization, IMS was incubated with
2
. Experimental
KpDERA (3 mg/mL) or buffer (50 mM TEA, pH 7.5), washed 4 times
2
.1. Reagents
◦
with buffer and once with ddH O, and dried at 60 C. The samples
2
◦
were degassed at 60 C for 24 h under vacuum.
IMS particles with pore size 20 nm (MACS P-20H) were devel-
Scanning electron microscope (SEM) images were obtained
using S-800 (Hitachi High-Technologies Corporation, Tokyo, Japan).
oped by JGC Catalysts and Chemicals Ltd., Kanagawa, Japan.
Acetaldehyde, DR5P, DL-G3P, ␣-glycerophosphate dehydrogenase-
triose phosphate isomerase from rabbit muscle (TPI/GDH), and
triethanolamine (TEA) were purchased from Sigma–Aldrich Japan
K.K. (Tokyo, Japan), the p-anisaldehyde (containing acetic acid and
H SO ) ethanol solution was obtained from Tokyo Chemical Indus-
2.4. Adsorption of KpDERA onto IMS
To study the adsorption of KpDERA onto IMS particles, a quantity
2
4
of IMS particles (50 mg) was incubated with 1 mL KpDERA solution
try Co., Ltd. (Tokyo, Japan). ␤-d-Thiogalactopyranoside (IPTG) and
nicotinamide adenine dinucleotide phosphate in the reduced form
◦
(
2 mg/mL) at 4 C for 24 h on a Rotator RT-50 (Taitec Corporation,
Saitama, Japan) to establish the adsorption equilibrium. The IMS
(
(
NADH) were purchased from Wako Pure Chemical Industries Ltd.
Osaka, Japan). All chemicals used in this study were high-quality
◦
was removed by centrifugation at 20,800 × g for 2 min at 4 C, and
the protein concentration in the supernatant was measured using
the BCA Protein Assay Reagent (Thermo Fisher Scientific Inc., Mas-
sachusetts, USA). The quantity of protein adsorbed was calculated
by subtracting the amount of protein in the supernatant after cen-
trifugation from the amount of KpDERA present before adsorption.
For the enzyme activity studies, 200 mg of IMS was incubated
with 3.5 mg KpDERA (1.4 mg/mL) in 50 mM TEA buffer (pH 7.5)
and analytical grade.
2.2. Subcloning and purification of KpDERA
The KpDERA gene [16] was amplified using the poly-
merase chain reaction (PCR) using the primers: forward,
5
ꢀ
ꢀ
ꢀ
◦
-GACATATGACTGATTTATCTGCAAGCAGCCTG-3 and reverse, 5 -
at 4 C on a Rotator RT-50. The IMS-immobilized KpDERA was
ꢀ
◦
AGACTCGAGTTAGTAGCTGCTGGCGCTCTTACC-3 . The PCR product
was ligated to the pBluescript II (SK+) plasmid (Agilent Tech-
nologies, Inc., California, USA) and sequenced to confirm its
veracity using the dideoxynucleotide chain termination method.
N-terminal hexahistidine-tagged KpDERA was produced by insert-
ing the appropriate clone into the NdeI–XhoI sites of the pET28b
vector (EMD Chemicals, Inc., California, USA).
collected by centrifugation at 2150 × g for 5 min at 4 C. The IMS-
immobilized KpDERA was washed five times then resuspended in
the buffer to a protein concentration of 1 mg/mL.
2.5. Enzyme activity
The DR5P cleavage activity was determined by measuring the
oxidation of NADH in a coupled assay using glycerol-3-phosphate
dehydrogenase and triose phosphate isomerase [17]. The assay
mixture (2 mL) contained 50 mM TEA (pH 7.5), 0.2 mM NADH, 1 mM
DR5P, 3 ␮L TPI/GDH, and 1.05 ␮g free or IMS-immobilized KpDERA.
The reduction in absorbance at 340 nm was monitored using a Shi-
mazu UV-2450 spectrophotometer (Shimazu, Kyoto, Japan). The
E. coli strain BL21 StarTM (DE3) (Invitrogen, California, USA)
was transformed with the KpDERA expression vector and grown in
Turbo broth (Athena Environmental Sciences, Inc., Maryland, USA)
at 37 C. The expression of KpDERA was induced by the addition of
IPTG to a final concentration of 1 mM. After incubation for a further
◦
◦
4
h at 37 C, the cells were harvested by centrifugation at 7500 × g
−
1
−1
for 5 min. The cell pellet was resuspended in 20 mM Tris–HCl
extinction coefficient of NADH was taken to be 6.22 mM cm .
(
pH 7.5), 500 mM NaCl containing 10 mM 2-mercaptoethanol, snap
The DR5P production activity was determined using a modifica-
tionofthe method reported byChenetal. [18]. Thereaction solution
(350 ␮L) contained 100 mM TEA (pH 7.5), 300 mM acetaldehyde,
100 mM DL-G3P, and 3 ␮g/mL free or IMS-immobilized KpDERA.
◦
frozen in liquid N , and stored at −80 C.
2
To purify KpDERA, the cells were thawed and 1 mM phenyl-
methylsulfonyl fluoride, 1 ␮g/mL pepstatin A, 0.5 mg/mL lysozyme,
1
After incubation at 4 C for 30 min, the cells were disrupted by
◦
mM MgCl , 100 units DNase1, and 10 mM imidazole were added.
The solution was incubated at 25 C for 20 min with shaking. Forty
2
◦
microliters of these mixtures were removed and centrifuged at
◦
◦
sonication and were centrifuged at 100,000 × g for 1 h at 4 C.
20,800 × g for 5 min at 4 C. A 20 ␮L aliquot of the supernatant was
The supernatant was then filtered through a 0.45 ␮m filter and
loaded onto a HisTrap HP column (GE Healthcare UK Ltd., Bucking-
hamshire, England) on the ÄKTA Explorer liquid chromatography
system (GE Healthcare UK Ltd.). The protein was eluted using a con-
quenched by addition of 8 ␮L 60% perchloric acid and was incu-
bated on ice for 10 min. This solution was neutralized with 13.4 ␮L
4 M NaOH and 179 ␮L 1 M TEA, pH 7.5. DR5P in the resulting super-
natant was measured by the cysteine-sulfate method [19]. One unit

T.Y. Nara et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 181–186
183
of enzyme activity refers to the activity required to produce or
degrade 1 ␮mole DR5P per minute.
2.6. Acetaldehyde tolerance of free and IMS-immobilized KpDERA
To investigate the enzyme aldehyde tolerance, 0.2 units of IMS-
immobilized KpDERA were incubated in a 50 mM TEA, pH 7.5,
◦
solution containing 300 mM acetaldehyde (200 ␮L) at 25 C for the
indicated times. The samples were washed with 50 mM TEA, pH 7.5,
and the DR5P cleavage activity was measured. In the case of the free
TM
enzyme, acetaldehyde was removed by filtration through a Zeba
Desalt Spin Column (Thermo Fisher Scientific Inc, Massachusetts,
USA).
2.7. Time course of DR5P production
The reaction mixture containing 0.2 units of free or IMS-
◦
immobilized enzyme was incubated at 25 C. Aliquots (25 ␮L) of
mixtures were removed at the indicated time points and cen-
◦
trifuged at 20,800 × g for 5 min at 4 C. Twenty microliters of the
supernatant were quenched by addition of 8 ␮L 60% perchloric acid
and neutralized with 13.4 ␮L 4 M NaOH and 179 ␮L 1 M TEA, pH 7.5.
2.8. Sequential aldol condensation
Fig. 1. SEM image of IMS particle. A high magnification image is shown in the inset.
The sequential aldol condensation reaction was analyzed by
thin-layer chromatography (TLC) using a modification of the
method described by Sakuraba et al. [20]. The reaction mixture
showed that IMS was a type IV mesoporous material according
to the International Union of Pure and Applied Chemistry (IUPAC)
classification system [22]. McBain described a “bottle-neck” theory,
which proposed that adsorption isotherms are determined by the
diameter of the inner pores, and desorption isotherms are deter-
mined by the pore openings [23]. According to this theory, the pore
diameters of IMS, calculated from the adsorption and desorption
isotherms, were 33 and 21 nm, respectively.
(
100 ␮L) containing 100 mM TEA (pH 7.5), 500 mM acetaldehyde,
and 0.5 mg/mL free or IMS-immobilized KpDERA was incubated at
◦
2
5 C for 24 h with shaking. An aliquot (4 ␮L) of the sample was
spotted onto a TLC plate (Silica gel 60 F254, Merck Ltd., Tokyo, Japan)
and developed with a running solvent consisting of 1-butanol,
acetic acid, and H O at a ratio of 4:1:1 (v/v/v). The reaction products
2
were localized using a p-anisaldehyde ethanol solution.
3.3. Immobilization of KpDERA on IMS
3
. Results and discussion
Fig. 3 shows the adsorption isotherm for KpDERA on IMS. The
3
.1. Expression and purification of KpDERA
The expression vector containing the N-terminal hexahistidine-
isotherm curve indicated that adsorption increased sharply then
reached a plateau. This type of isotherm can be modeled using
the Langmuir equation [24]. The maximum adsorption capacity,
tagged KpDERA gene was transformed into the E. coli strain BL21
TM
Star
(DE3), and expression of KpDERA was induced. The pro-
tein was purified through a HisTrap HP column. The protein was
purified to near homogeneity and was found to be suitable for
subsequent studies (Supplementary Fig. 1). The final yield of the
purified KpDERA was 60 mg/L E. coli culture. Gel filtration anal-
ysis (Supplementary Fig. 2) revealed that the molecular mass of
KpDERA was 58 kDa. The molecular mass of KpDERA, determined
by SDS-PAGE analysis (Supplementary Fig. 1) and predicted from
the amino acid sequence [16], was 28 kDa. Therefore, KpDERA was
confirmed to be present as a dimer. It has been reported that E. coli
DERA (EcDERA) consists of two identical 28 kDa subunits with unit
cell dimensions 49 A˚ × 42 A˚ × 145 A˚ [21]. The amino acid sequence
of KpDERA is similar to that of EcDERA (86% identity) [16]. The unit
cell dimensions of KpDERA may also be similar to those of EcDERA.
3.2. Characterization of IMS
As shown in Fig. 1, the IMS particles were characterized as well-
ordered spheres. Dynamic light scattering analysis revealed that
the mean diameter was 3 ␮m (data not shown). A magnified image
Fig. 2. Nitrogen adsorption/desorption isotherms and pore size distributions of the
IMS particles. Nitrogen adsorption (ꢀ) and desorption (ꢁ) isotherms. (Inset) Pore
size distributions: adsorption (ꢀ), desorption (ꢁ). The isotherms were measured
using an AUTOSORB-1 analyzer. The pore size distributions were determined by the
BJH method using the adsorption and desorption branches.
(
Fig. 1 inset) shows that the IMS particles contained pores formed
from the voids between the aggregated silica nanoparticles.
Fig. 2 shows the nitrogen adsorption/desorption isotherms and
the pore size distributions of the IMS particles. The isotherm curves

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Fig. 3. Adsorption isotherm of KpDERA on IMS. KpDERA (1 mL, varying concentra-
tions) was permitted to adsorb onto 20 mg IMS particles for 24 h at 4 C. The protein
concentration in the supernatant was subsequently measured.
Fig. 5. Acetaldehyde tolerance of free and IMS-immobilized KpDERA. Free (ꢀ) or
IMS-immobilized (ꢂ) KpDERA (0.2 units) were incubated with 300 mM acetalde-
◦
◦
hyde at 4 C for the indicated times, and DR5P cleavage activity was subsequently
measured. The relative activity was calculated with respect to the activity of each
sample at 0 min.
calculated by curve fitting, was 39.7 mg/g support. Because the
pore openings in the IMS particles were sufficiently larger than
the unit cell dimensions of KpDERA (estimated from the EcDERA
structure), it was possible that KpDERA was immobilized within
the pores of IMS. To confirm that KpDERA was present within the
pores of IMS, the nitrogen adsorption before and after KpDERA load-
ing was investigated. As shown in Fig. 4, the magnitudes of the
nitrogen adsorption and pore size distribution curves decreased
after adsorption of KpDERA. The surface area and total pore volume
in the supernatant of the wash solutions during the immobilization
step or in the reaction mixtures (data not shown). Enzyme confor-
mational changes and/or blocking of the active site may have been
induced by the enzyme–support interaction [26]. Limited substrate
diffusion and steric hindrance may also have decreased the activity
of the immobilized KpDERA relative to that of the free enzyme [27].
2
3
also decreased (41.4–29.9 m /g and 0.211–0.171 cm /g, respec-
tively). These results suggested that the IMS pores were occupied
by KpDERA [25].
3.5. Acetaldehyde tolerance of free and IMS-immobilized KpDERA
DERA has shown poor tolerance to high aldehyde concentra-
tions, and large quantities of enzyme are required to obtain useful
product yields [10]. This may limit its practicality for industrial
applications. The resistance of DERA to aldehydes is, therefore,
required. To investigate the effects of immobilization on acetalde-
hyde tolerance, free or IMS-immobilized KpDERA was incubated in
a buffered solution containing 300 mM acetaldehyde, and the DR5P
cleavage activity was measured. As shown in Fig. 5, the activity of
the free enzyme decreased to 20% after incubation for 30 min, and
the activity disappeared completely after 90 min. In contrast, IMS-
immobilized KpDERA retained more than 50% of its activity after
3.4. Activities of free and IMS-immobilized KpDERA
The specific activities of free and IMS-immobilized KpDERA for
the DR5P cleavage reactions were 119 and 18.6 units/mg, respec-
tively. Indeed, the specific activity decreased to about 16% by
immobilization on IMS. Several factors may account for the reduc-
tion in the specific activity of IMS-immobilized KpDERA. Enzyme
leaching was improbable because the proteins were not observed
3
0 min, and showed 11% activity after 90 min. This result indicated
that the acetaldehyde tolerance was improved by immobilization
within the pores of IMS.
Enzyme inactivation by aldehydes arises mainly from Schiff base
formation between the surface lysine residues and the aldehydes
[
28,29]. Because the silica support surface is negatively charged at
pH > 2 [30], positively charged lysine residues of KpDERA may be
masked from acetaldehyde attack by interacting with the IMS. It is
also suggest that the enzyme structure was stabilized by confine-
ment in a narrow space, even if the aldehyde modified the enzyme
surface.
Chemical modification of protein surface is also a useful tech-
nique for stabilizing enzymes [31–33]. Chemical modification with
formaldehyde causes the transformation of primary amine groups
on the enzyme surface into secondary amino groups [34,35].
This modification might prevent deleterious modification effect of
acetaldehyde on KpDERA surface. Furthermore, when penicillin G
acylase was immobilized through multipoint attachment prior to
chemical modification, the enzyme showed superior resistance to
the effect of chemical modification with formaldehyde [34]. Mul-
tipoint covalent attachment, in which the enzyme is immobilized
by contact with the support through multipoint covalent bonds,
highly stabilizes the enzyme by rigidification of enzyme struc-
ture [31,36–38]. Silica surface contains very dense silanol groups
Fig. 4. Changes in the nitrogen adsorption isotherms and pore size distributions of
the IMS particles before and after immobilization of KpDERA. The IMS particles were
incubated in buffer (ꢀ) or KpDERA in buffer (ꢂ). The isotherms were then measured
using an AUTOSORB-1 analyzer. The pore size distributions were determined by the
BJH method using the adsorption branches.
2
(4.3–6.7 OH/nm ) [39] that can be easily functionalized with the

T.Y. Nara et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 181–186
185
Fig. 6. Time course of DR5P production. Free (ꢀ) or IMS-immobilized (ꢂ) KpDERA
0.2 units) was incubated in a reaction mixture containing 300 mM acetaldehyde
(
◦
and 100 mM DL-G3P at 23 C for the indicated times. The concentration of DR5P
was subsequently measured.
functional groups for covalent immobilization of enzymes. There-
fore, the functionalized IMS could also be used to stabilize the
enzyme through multipoint attachment.
3.6. Aldol condensation using a natural substrate of DERA
Fig. 7. TLC analysis of the sequential aldol condensation product. Lane 1, buffer con-
trol; Lane 2, free; Lane 3, IMS-immobilized KpDERA. The reaction mixture containing
5
00 mM acetaldehyde was incubated with 0.5 mg/mL free or IMS-immobilized
The aldol condensation reaction catalyzed by DERA is impor-
◦
KpDERA at 25 C for 24 h. The products were developed on TLC plates and were
tant for organic synthesis. However, quantification of the sequential
aldol condensation products is difficult. We evaluated these prod-
ucts by analyzing the production of DR5P, which is the natural
product of DERA, from G3P in the buffered solution containing
stained using a p-anisaldehyde ethanol solution.
4. Conclusions
3
00 mM acetaldehyde.
The specific activity of the IMS-immobilized KpDERA, surpris-
KpDERA was successfully immobilized on IMS particles, which
are aggregate mesostructures formed from the hexagonal close
packing of silica nanoparticles. The immobilized enzyme exhibited
superior aldehyde tolerance relative to the free enzyme. The equi-
librium concentration of the sequential aldol condensation product
was higher when produced by the IMS-immobilized KpDERA. These
results suggest the potential use of IMS as an immobilization sup-
port for DERA for the production of pharmaceutically important
intermediates.
ingly, displayed 74% activity relative to that of the free enzyme
75.4 versus 102 units/mg). Fig. 6 shows the time course of DR5P
(
production for a fixed initial quantity of enzyme units. The DR5P
concentration reached a plateau after 10.5 h reaction with the free
enzyme, whereas the IMS-immobilized KpDERA retained activity
after 20 h. The enhanced activity of the immobilized enzyme may
indicate improved acetaldehyde tolerance. It is also possible that
the equilibrium of the enzyme reaction shifted to favor the aldol
condensation product.
Acknowledgments
The authors thank Mr. Shuzo Kojima and Dr. Naoki Tahara
and (JGC Corporation) for their helpful discussions concerning the
experimental design and results. This work was performed under a
collaboration with the JGC Corporation and JGC Catalysis and Chem-
icals Ltd. entitled “Development of enzyme reaction systems using
the new immobilization support”.
3
.7. Sequential aldol condensation
Finally, the sequential aldol condensation reaction with
acetaldehyde was analyzed by TLC analysis. Fig. 7 showed that
the TLC spot intensity corresponding to the aldol condensation
products produced from 500 mM acetaldehyde was higher for the
IMS-immobilized KpDERA than for the free enzyme. This result
suggested that the efficiency of DERA as a catalyst for sequential
aldol condensation was also improved by immobilization on IMS.
It should be noted that it was not possible to identify the physical
properties of this product, although the TLC pattern was similar to
that reported by Sakuraba et al. [20]. Further studies are required
to determine whether this product was a chiral trideoxyhexose.
Previous attempts to improve the yield of the industrially useful
chiral trideoxyhexoses have used DERA from a variety of organisms
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2010.10.008.
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