Journal of Molecular Catalysis B: Enzymatic 8586 (2013) 220228

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Journal of Molecular Catalysis B: Enzymatic

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Immobilization of glucose 6-phosphate dehydrogenase in silica-based hydrogels:

A comparative study
S. Cumana a, , J. Simons b , A. Liese b , L. Hilterhaus b , I. Smirnova a
a
b

Institute of Thermal Separation Processes, Hamburg University of Technology, Eiendorferstr. 38, D-21073 Hamburg, Germany
Institute of Technical Biocatalysis, Hamburg University of Technology, Denickestr. 15, D-21073 Hamburg, Germany

a r t i c l e

i n f o

Article history:
Received 9 May 2012
Received in revised form
17 September 2012
Accepted 17 September 2012
Available online 25 September 2012
Keywords:
Enzyme immobilization
Encapsulation
Dehydrogenase
Solgel
Hydrogels

a b s t r a c t
Glucose 6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides catalyzes the oxidation of
glucose-6-phosphate (G6P) to 6-phosphogluconate in the presence of NAD(P)+ . This enzyme is part of a
multistep synthetic pathway for the production of hydrogen from starch and water under mild conditions.
The encapsulation of the enzyme in silica-based matrices should protect the enzyme from denaturation
and serve as a rst step in the development of silica-based matrices for the encapsulation of further
enzymes required in the reaction system. This base formulation must be adapted to the corresponding
enzyme. In this work the solgel method was used for the encapsulation of G6PDH in silica-gels prepared
by different methods. The objective was to determine the approach which offers the highest enzymatic
activity of G6PDH after encapsulation. Different routes based on alkoxysilanes, aqueous and ethylene
glycol modied silanes as silica precursors were investigated. To validate the activity and stability of the
encapsulated enzyme, a comparison to covalently immobilized G6PDH on amino functionalized particles
via glutaraldehyde was carried out. The nal goal of this project is to use the enzyme-doped gels in owthrough microreactor systems, where single or more reaction steps can be carried out in sequence. G6PDH
is the rst of the 13 enzymes which must be studied.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Enzymes are biological catalysts, which will play an important
role in industrial scale processes since they are versatile, regio-,
chemo- and enantioselective [1]. Since the isolation of enzymes is
an expensive procedure, the reusability of enzymes is particularly
for industrial applications a signicant demand. Typical immobilization techniques are physical adsorption, immobilization via
ionic interactions, covalent binding and encapsulation into polymeric gels. During encapsulation the enzyme is enclosed by a
polymeric matrix which protects it from aggregation and denaturation by unfolding [2]. Therefore it is not necessary to attach
the enzyme molecules to the gel walls by adsorption or other ionic
or covalent bonds. These interactions could affect negatively the
performance of the enzymes [2,3].
This work focuses on the immobilization of G6PDH from Leuconostoc mesenteroides, by encapsulation in mesoporous silica-gels
via the solgel method and its comparison to covalent immobilization. G6PDH converts glucose-6-phosphate to 6-phosphogluconate
in the presence of NAD(P)+ and is part of a synthetic pathway for

Corresponding author. Tel.: +49 40 42878 3429; fax: +49 40 42878 4072.
E-mail address: sucre.cumana@tu-harburg.de (S. Cumana).
1381-1177/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.09.014

the high-yield hydrogen production from starch and water developed by Zhang et al. [4,5]. This reaction system presents a hydrogen
production from abundant renewable biomass and achieves zero
net greenhouse gas emissions. This synthetic reaction sequence
involves 13 enzymes derived from different organisms and could
produce 12 mol H2 /mol glucose. This yield is three times higher
than in natural bioconversion processes for biological production of
H2 . Since feedback inhibition makes this process inefcient when
executed in one pot, this work focuses on the concept for multistep biocatalysis. This synthetic enzymatic pathway would open
a new way to efciently produce H2 from inexpensive renewable
biomass, e.g. cellulose [6]. Principally it would be possible to conduct the reaction in a corresponding microreactor. However, in a
rst step a suitable encapsulation technique allowing the multistep
catalysis should be established. The requirements on the encapsulation are a high enzymatic activity upon encapsulation, reusability
of the enzyme-doped gels and thus a long-term stability of G6PDH.
The demands on the silica gel are suitable mechanical stability,
a high surface area per volume ratio, adequate porosity and simplicity of synthesis. In this work different solgel approaches such
as aqueous, alkoxy and modied silane routes are presented. The
strong dependence between the silica precursor used for the encapsulation and the resulting enzymatic activity has already been
demonstrated [7]. Additionally different aging media and their

S. Cumana et al. / Journal of Molecular Catalysis B: Enzymatic 8586 (2013) 220228

inuence on the gel properties and activity of the encapsulated

enzyme are examined. Further on the solgel approach is compared
to the immobilization of G6PDH on amino functionalized particles using glutaraldehyde. G6PDH has already been immobilized
and co-immobilized with other enzymes on amino functionalized
particles [8].
2. Experimental

221

HCl were mixed, as described above, and the mixture was vigorously stirred for 15 min. A higher water content was used in order
to dilute the methanol generated during the hydrolysis reaction
(1:17:2 102 molar in TMOS:H2 O:HCl). The diluted sol was then
cooled as previously described. In the second step NH4 OH was
added to the buffered enzyme solution to a nal pH value of approximately 78. Due to the higher pH value, the gelation occurred
within 30 s. Both types of TMOS-derived gel were aged in TrisHCl
at 2.5 C.

2.1. Solgel encapsulation of G6PDH

The encapsulation of G6PDH has been performed with different precursors: the alkoxysilanes tetraethyl orthosilicate (TEOS,
Merck KGaA) and tetramethyl orthosilicate (TMOS, Fluka), colloidal silica (LUDOX HS-40, Aldrich) and the water soluble silica
precursor ethylene glycol modied silane (EGMS). The precursors were used as received without further purication. EGMS
was prepared from TEOS and ethylene glycol based on the work
of Mehrotra et al. [912]. The detailed experimental procedures
are described below. Glucose 6-phosphate dehydrogenase from L.
mesenteroides was purchased from SigmaAldrich as lyophilized
powder (700 Units mgprotein 1 ) and dissolved in TrisHCl buffer
100 mM pH 7.5 (Roth). The concentration of the resulting enzyme
solution was 3.75 mg/ml. In every case the gels had a volume of
171 l and were prepared by pouring the sol solution in mould
syringes of 2 ml. The G6PDH loading of each gel was 0.9 gG6PDH .
2.1.1. TEOS-derived gel
Mesoporous silica monoliths were prepared using a modication of an existent method based on the solgel process [13]. TEOS,
deionized water and hydrochloric acid (Merck KGaA) were mixed
and stirred vigorously at room temperature for 60 min. Additional
water was added to the sol and the solution was stirred for further
15 min until the pH value of the homogeneous sol was between 3
and 4. The achieved molar ratios of TEOS:H2 O:HCl were 1:25:102 .
Since the ethanol generated during the hydrolysis reaction may
worsen enzymatic activity, it was partially evaporated from the sol
under vacuum [3,14,15]. The removal of the alcohol was achieved
with a Rotavapor (RE111 Bchi Labortechnik AG, Switzerland)
under vacuum at 42 C using different evaporation times: 12.5 and
25 min. The sol was then left to cool down at 2.5 C for at least
1 h. For comparison some samples were not subjected to alcohol
evaporation. In order to obtain gel densities similar to those where
ethanol was evaporated additional water was added.
For the encapsulation the hydrolyzed TEOSsol was mixed in
a volume ratio 1:1 with a basic solution, consisting of NH4 OH,
TrisHCl and diluted G6PDH (dilution factor 1:100) in volume ratios
of 1:13.3:5.6 respectively. Gelation occurred within 2 min at pH
8. Ten minutes after gelation the samples were placed into falcon
tubes lled with the aging medium and stored at 2.5 C. Some of the
samples were aged by immersion in pure TEOS and other samples
were aged in TrisHCl 100 mM.
2.1.2. TMOS-derived gel
TMOS-derived sol stock solutions were prepared by mixing
TMOS, deionized water and HCl at room temperature at molar
ratios of 1:4:2 102 in TMOS:H2 O:HCl respectively. The mixture
was stirred vigorously for 30 min and the clear sol was left to cool
down at 2.5 C for about 1 h before encapsulation. The hydrolyzed
TMOSsol was then mixed with an equal volume of a buffered solution including the diluted enzyme solution (volume ratios of 1:2.5
in TrisHCl and diluted G6PDH respectively). Gelation occurred
within 1 min at a pH value of 6. The 171 l loaded gels were left
for aging in TrisHCl at 2.5 C.
A second TMOS-derived type of gel was synthesized with the
approach that follows. TMOS, distilled and deionized water and

2.1.3. Aqueous gel

Aqueous sol stock solution was prepared by a modication of a
solgel process published elsewhere [16]. Colloidal silica LUDOX
(40 wt%), deionized water and HCl were stirred at room temperature at respective mass ratios of 1:0.3:3 102 . Afterwards a
basic solution containing 3-aminopropyltriethoxysilane (APTES),
the diluted enzyme solution and TrisHCl at respective volume
ratios of 1:14:35 was prepared. The basic solution was mixed with
an equal volume of the clear sol and gelation occurred within
12 min. The gel turned opalescent after gelation. The nal pH value
was 8. Ten minutes after gelation, the samples were immersed into
the aging medium and stored at 2.5 C. TrisHCl pH 9.5 and TEOS
were used as aging media.
2.1.4. EGMS-derived gel
For the preparation of mesoporous gels EGMS and TrisHCl 7.5
were stored at temperature of 2.5 C for at least 1 h. They were
then mixed at a volumetric ratio of 1:5.3 and stirred for 30 s. Finally
the diluted enzyme solution was added to the mixture and stirred
for additional 30 s. Gelation occurred within 10 min and then the
samples were left for aging.
2.2. Covalent immobilization of G6PDH on amino functionalized
particles
20 mg Sepabeads EC-HA or Trisoperl amino particles were activated with 3 ml 260 mM glutaraldehyde for at least 70 min shaking
at room temperature. After washing the particles ve times with
3 ml double distilled water, G6PDH was immobilized in 100 mM
TrisHCl buffer pH 7.5 shaking over night at 4 C. Activity assays
were carried out of the wash fractions as well as of the immobilized
enzyme to check on complete immobilization.
2.3. Silica-gel properties
2.3.1. Pore size distribution and surface area
The pore size distribution and surface area of mesoporous gels
were determined via the nitrogen adsorptiondesorption method,
according to the BET and BJH theory. A Nova 3000e surface area and
pore analyzer (Quantachrome Instruments, Germany) was used for
the measurements.
Since N2 -adsorption cannot be applied to wet samples the measurement of the pore size and the surface area was performed
after supercritical drying of the gels. It is generally assumed that
the properties of the gels before and after supercritical drying do
not vary substantially [17,18]. To carry out the supercritical drying
the liquid inside the pores was exchanged for ethanol during 24 h.
Afterwards the wet gels were lled in an autoclave and drying was
performed with supercritical CO2 at 100 bar and 40 C for several
hours [19,20].
2.3.2. Shrinkage
The shrinkage of gel samples was measured by using a caliper.
Since the samples were synthesized in syringes, the gels have a
cylindrical shape. Thus the volume of the samples was easily determined by measuring their height and diameter. The measurement

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S. Cumana et al. / Journal of Molecular Catalysis B: Enzymatic 8586 (2013) 220228

of the volume of the wet gels started nearly 1 h after synthesis and
continued for four days. The gels were immersed in different aging
media, to investigate their shrinking behavior.
2.4. Activity measurements
The enzymatic activity of the G6PDH was obtained by activity
assays and kinetic studies. In both cases the activity measurements
were done for immobilized as well as for free enzymes. In this work
the relative activity has been dened as the specic activity of the
encapsulated enzyme divided by the specic activity of the free
enzyme at the same conditions.

After storage, the gel samples were cut into halves and placed
in 2 ml of 20 mM TrisHCl pH 7.5. The gels were cut into halves,
since otherwise they would not have t into the Eppendorf tubes
used for the tests. The samples were brought to 37 C at a shaking
speed of 200 rpm (model MHR 13, HLC Biotech, Germany). After
1 h the gel samples were removed and the protein content of the
remaining liquid was determined. Due to the very low concentration of enzyme used a linearization of the Bradford protein assay
according to Zor and Selinger was preferred [21]. This linearization
increases the accuracy and improves the sensitivity of the method
about 10-fold.
3. Results and discussion

2.4.1. Activity assays

The reagents were mixed in a UV cuvette and equilibrated to
37 C. The concentrations of the chemicals in the reaction mixture
were: 84 mM TrisHCl, 2.7 mM G6P, 0.67 mM NADP+ , 40 mM MgCl2
and 0.19 gG6PDH ml1 . For the assay with free enzyme, the reaction was initiated by adding enzyme solution to the reaction system
and the increase in absorbance due to the formation of NADPH was
recorded for 5 min. The absorbance was measured at 340 nm with
an Evolution 300 UV-Vis Spectrophotometer (Thermo Scientic,
Germany).
The activity measurements of encapsulated G6PDH were performed in two different ways. In the rst case, the gel was crushed
in the assay solution and in the second case the gel was not broken but put into a basket immersed into the assay solution. The
basket was rotated at approximately 120 rpm. The assay solution
was equilibrated to 37 C and the reaction was initiated by adding
the substrate (G6P). Samples were taken and the increment in
absorbance was recorded for approximately 5 min, corresponding
to the linear region of the absorbance dependency. All activity measurements were performed after one day of encapsulation, except
during long term experiments, in which the samples were aged for
4 or 37 days.
The activity of G6PDH immobilized on particles was determined
in 3 ml volumes. Immobilized G6PDH was incubated at 30 C while
shaking and the absorbance was recorded in desired time intervals. For repetitive batch experiments, G6PDH immobilized on
Sepabeads EC-HA were washed after activity measurements and
stored in 100 mM TrisHCl buffer pH 7.4 at 4 C.
2.4.2. Kinetic studies
The MichaelisMenten constant (KM ), the maximal reaction
rate (vmax ) and the turnover number (kcat ) were determined for
free and encapsulated enzymes. The initial rate measurements at
varying substrate concentrations were performed according to the
enzymatic assay described. In the experiments with encapsulated
enzymes the gels were not crushed.
The concentrations of G6P used were in the range of 0.023 mM,
while maintaining the concentration of NADP+ at a constant value
of 20 mM. The concentration of NADP+ was kept high in comparison to the KM value in order to use the MichaelisMenten equation
for a single substrate. The initial concentration of G6PDH in solution and in the encapsulated state was 0.19 g ml1 . The enzymatic
assay also contained 84 mM TrisHCl pH 7.5 and 40 mM MgCl2 . The
kinetic parameters KM and vmax were determined by means of a
non-linear regression analysis of the data.
2.4.3. Leaching tests
The leaching experiments were performed only with TEOS-gels
after 24 h storage at 2.5 C. The concentration of enzyme used was
increased to 0.21 mgG6PDH mlgel 1 , otherwise the protein content
would have been under the limit of detection of the methods
available.

In this work different silica precursors have been used to immobilize glucose 6-phosphate dehydrogenase by means of the solgel
process. The success of the encapsulation depends on several factors, which are discussed below.
3.1. Pore size distribution and surface area
The pore size distribution of the gels used for the encapsulation of enzymes must meet certain demands. The pores must
be large enough to ensure the transport of substrate and product
molecules within the porous network, but at the same time they
must be small enough to prevent leakage of encapsulated enzymes
[1,22,23]. If the walls of a microsystem were directly used for the
enzyme immobilization, the enzymatic activity might be limited
since the surface-to-volume ratio of a microchannel is rather small.
For instance in a 100 l cylindrical microchannel with an inner
diameter of approximately 1 mm and a length of 100 mm, the surface area available for immobilization would be nearly 104 m2 . On
the other hand, if the microchannels were lled with a monolithic
gel of a high density, for instance 0.3 g cm3 , and a specic surface
area of 300 m2 g1 , the surface area available for the immobilization would increase till almost 10 m2 . Therefore, monoliths were
proven the favorite supports to immobilize enzymes on microchips.
In order to increase enzyme loading capacity the surface area of the
gel has to be as large as possible [24]. Furthermore, a narrow pore
size distribution is desirable. The properties of the different gels are
summarized in Table 1.
3.1.1. TEOS-based gels
The pore size distribution of the TEOS-gels, produced after different times of ethanol evaporation and those aged in different
aging media were very similar (Fig. 1). The gure shows that the
gels had pores in the range of 3130 nm. Since the largest dimension of the enzyme molecule is 11.2 nm [25], the gels-based on TEOS
possess a wide range of pores where G6PDH can be immobilized.
The amount of pores smaller than the enzyme molecule, where the
enzyme cannot be immobilized, is small in comparison with the
rest of the pores of the gel. The results suggest that the evaporation
of the hydrolyzed TEOS sol does not have a signicant inuence on
the pore structure. Moreover, the surface areas of TEOS-gels aged in
TrisHCl were not signicantly different from those aged in TEOS. In
a previous work the surface areas of TEOS-based gels after supercritical drying were in the range of 6501000 m2 g1 depending
on the gel density and aging temperature [26]. The surface areas
obtained in this work for TEOS-gels were also in this range.
3.1.2. TMOS-based gels
The pores of TMOS-based gels (Fig. 1) were in the range of
4200 nm. The pore size distribution of the TMOS-derived gels
shifted to larger diameters and was much narrower than for

S. Cumana et al. / Journal of Molecular Catalysis B: Enzymatic 8586 (2013) 220228

223

Table 1
Pore structure parameter of different types of gels; surface area (BET-method), pore volume and pore diameter (BJH-method).
Type of gel

Aging medium

Surface area
(m2 g1 )

TEOS 25 min evaporation

TEOS No evaporation
TEOS No evaporation
TMOS
LUDOX
LUDOX
EGMS
EGMS

TEOS
TEOS
TrisHCl pH = 7.5
TrisHCl pH = 7.5
EtOH
TrisHCl pH 9.5
EtOH
TEOS

652
790
720
774
178
163
732
400

43
31
82
24
11
6
14
25

TEOS-based gels. This behavior has been observed previously in

the literature [27,28].
3.1.3. Aqueous gels
Compared to the transparent alkoxysilane-derived gels, the
monoliths prepared with LUDOX were opalescent. The pore size
distribution was in the range of 4130 nm (Fig. 1). Aqueous gels had
much lower pore volume than alkoxysilane-based gels. By aging the
gels in TrisHCl pH 9.5 instead of ethanol the surface area slightly
decreased, but larger pore volume and narrower pore size distributions were reached. Although the pore morphology could be
improved by aging in TrisHCl, the pore size distribution in the
range of 20200 nm might still be too large respect to the enzyme
size and lead to leaching.
3.1.4. EGMS-based gels
The pore sizes of EGMS-based gels ranged from 4 to 140 nm. The
distribution in gels stored in EtOH was narrower than in gels stored
in TEOS (Fig. 1) and both appear suitable for encapsulation.
A possible explanation for the larger pores, lower specic surface areas and lower pore volumes obtained after aging in TEOS may
be the Ostwald ripening. It means that the diffusion of monomers
from smaller to larger primary silica particles is enhanced, due to
the greater solubility of the single monomer molecules in the larger
monomer droplets. Silica particles precipitate into regions of negative curvature onto the structure network of the gel. Therefore the

(a)

4.0

dV(logd)

2.59
2.12
2.57
5.25
0.44
1.35
2.61
1.11

Pore diameter (nm)

0.17
0.10
0.08
0.22
0.01
0.07
0.04
0.18

15.6
6.3
13.9
21.2
4.0
26.0
13.0
4.0

2.5
4.4
2.2
7.5
0.1
0.3
1.7
0.5

necks between the particles grow and small pores are lled. As a
result the pore size of the gel increases, while the specic surface
area and pore volume decrease [29].
The results show that the surface area and pore size distribution
of gels depend not only on the precursor used but also on the aging
medium. Gels based on alkoxysilanes possess smaller pores, narrower pore size distribution and higher surface areas than aqueous
ones. These characteristics are positive in terms of enzyme loading and leaching behavior, therefore alkoxysilanes show potential
for G6PDH encapsulation. TMOS is commonly used for two reasons.
Firstly, because its rate of hydrolysis and polycondensation is much
faster than for TEOS. Secondly, because the methanol generated
during the solgel reaction has a polarity much similar to water
than the other alcohols from the same family and should cause
less denaturation of the encapsulated protein [30]. Since LUDOX
and EGMS-gels do not involve alcohol, they constitute an enormous
potential for the encapsulation of the enzyme without important
activity losses.
3.2. Shrinkage
Shrinkage is an important problem when dealing with solgel
processes. TEOS-gels were aged in the silica precursor (TEOS) and in
TrisHCl buffer pH 7.5. TMOS-gels were stored in the buffer solution only. The shrinkage of aqueous gels could not be measured,
since they were too weak and consequently were broken by the
18.0

4.5

(b)

16.0

3.5

14.0

3.0

12.0

2.5

10.0

2.0

8.0

1.5

6.0

1.0

4.0

0.5

2.0

0.0

0.0
2

20

200

1.8

20

200

6.0

(c)

1.6
1.4

dV(logd)

Pore volume
(cm3 g1 )

1.2

(d)

5.0
4.0

1.0

3.0

0.8

2.0

0.6
0.4

1.0

0.2

0.0

0.0
2

20

Pore Diameter (nm)

200

20

200

Pore Diameter (nm)

Fig. 1. Pore size distribution of: (a) TEOS gels aged in TEOS with no evaporation () and after 25 min () evaporation, (b) TEOS () and TMOS () gels aged in TrisHCl pH
7.5, (c) LUDOX gels aged in EtOH () and in TrisHCl pH 9.5 () and (d) EGMS gels aged in TEOS () and in EtOH ().

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S. Cumana et al. / Journal of Molecular Catalysis B: Enzymatic 8586 (2013) 220228

Activity (Units mg-1)

60

50
40
30

20
10

12.5

25

Evaporation Time (min)

Fig. 2. Shrinkage of silica-based gels in different aging media during 4 days of aging:
TEOS-gels in TrisHCl (gray), TEOS-gels in TEOS (dotted), EGMS-gels in EtOH (white),
EGMS-gels in TEOS (striped) and TMOS in TrisHCl (black).

Fig. 3. Specic activity of free G6PDH (white) and of G6PDH encapsulated in TEOSderived gels depending on the evaporation time of the sol (gray).

caliper. Finnie et al. reported that minimal shrinkage of colloidal

silica-derived gels was observed during aging [16].
The results prove that shrinkage is not only affected by the precursor but also by the aging medium (Fig. 2). The most pronounced
volume decrease occurred one day after the gel point. The results
show that TEOS-gels shrink more than TMOS-gels during aging in
TrisHCl. It was also found that aging in the silica precursor helps
to reduce shrinkage. Similar results regarding the better mechanical properties of gels aged in TEOS were obtained by Hwang et al.
[31]. They observed a densication of the structure upon aging in
TEOS/ethanol solutions which was more pronounced for higher
concentration of the precursor.
Since the gels were immersed in a liquid no evaporation could
have taken place, and hence the shrinkage must be originated from
the aging process only. The aging medium has a signicant inuence on the shrinkage of the gel network [26]. While the storage
in TrisHCl leads to dissolution and reprecipitation of silica from
the original gel network, the storage in TEOS entails the dissolution and reprecipitation also from the silica solution. It means that
aging in TEOS leads to a higher stiffness and mechanical strength of
the silica matrix and as a consequence reduces gel shrinkage [32].
Volumetric contraction of the monolithic gels disables their use in
columns or microuidic systems and must therefore be avoided.
EGMS-gels were aged in TEOS and ethanol. No signicant shrinkage occurred after 4 days of aging (Fig. 2). After 3 days of aging, the
shrinkage observed was less than 5% in either medium. EGMS-gels
aged in TEOS shrunk less than those aged in ethanol. This tendency
is similar to the one observed for alkoxysilane-gels, in which aging
in TEOS led to a lower shrinkage.
The results show that syneresis occurs and it depends on the silica precursor as well as on the aging medium. Gels prepared by the
aqueous route were not mechanically stable enough and shrinking
cannot be determined as they break. Gels based on alkoxysilanes
were more resistant than aqueous ones. Their mechanical properties can be improved by aging them in the silica precursor instead
of in buffer solution. In this way the possibility to use the gels in
ow-through systems arises.

It was not possible to determine immobilization yield due to

two main reasons: rst, the low quantities of protein used made
the common methods of protein determination inapplicable; second, an activity assay from the leached enzyme would not consider
deactivation by the encapsulation method. In both cases the consequence would be an underestimation of the quantity of protein
leached and of the activity of the remaining encapsulated enzyme.
Since the surface area of the gels used in this work is very high and
the enzyme molecules are greatly dispersed, it is assumed that the
enzymatic activity in the crushed gels is equivalent to the activity
of the total enzyme immobilized. This afrmation also supposes
that possible differences in the particle size of the broken gel do
not strongly inuence the activity values. In this way the intrinsic
activity losses from the encapsulation method are also pondered.

3.3. Activity of immobilized glucose 6-phosphate dehydrogenase

Immobilization may lead to losses of enzymatic activity. Thus
different solgel approaches were tested in order to nd the one
which preserves the maximum of activity of G6PDH after encapsulation. First the activity after encapsulation into different gels
aged in different media was measured. Later the activity of G6PDH
encapsulated in selected gels was determined at different aging
times. In this part the activity measurements were performed by
crushing the gels after one day of aging as described in Section 2.

3.3.1. TEOS-based gels

After entrapment of proteins within alkoxysilane-derived materials a decrease in the activity of the encapsulated enzyme with
time is generally observed. It has been attributed to the presence
of relatively high levels of alcohol byproducts from the hydrolysis
reaction during the solgel process [14]. During the hydrolysis of
TEOS and TMOS ethanol and methanol are respectively produced.
Several approaches to avoid the presence of alcohol are possible,
including the substitution of alkoxysilanes by aqueous precursors
or by evaporating the alcohol generated during the solgel reaction.
The rst approach used in this work for enzyme encapsulation was a modication of the solgel route reported by Ferrer
et al. for its simplicity [13]. The method is based on the gentle
vacuum elimination of the alcohol by rotavaporization methods.
The experiments aimed at the investigation of the dependence of
the concentration of residual ethanol in the gel on the activity of
G6PDH. Vacuum evaporation of ethanol in the hydrolyzed sol was
performed for 12.5 and 25 min and the enzymatic activity was compared with samples not subjected to ethanol removal. Contrary to
the expectations, no important enhancement of enzyme activity
could be achieved by removal of ethanol (Fig. 3). The concentration
of ethanol remaining in the sol has been estimated as 0.44, 1.08
and 2.95 M for 25 min, 12.5 min and no evaporation respectively.
The results indicate that encapsulation of G6PDH stabilizes the
enzymes against denaturation caused by ethanol. Similar results
have been reported for the encapsulation of catalase where the silica gel stabilized the enzyme to higher methanol concentrations
[33].
The relative activities for TrisHCl and TEOS aged gels are presented in Fig. 4. Surprisingly G6PDH aged in buffer solution retained
less than 42% of its activity, while in gels aged in TEOS the activities
were similar to those of the free enzyme.

S. Cumana et al. / Journal of Molecular Catalysis B: Enzymatic 8586 (2013) 220228

Fig. 4. Comparison of activities of encapsulated G6PDH in different gels aged in

different media: TrisHCl pH 7.5 (white), TrisHCl pH 6 (black), mother liquor (gray)
and TEOS (striped).

3.3.2. TMOS-based gels

Since methanol is less harmful for enzyme and cells than ethanol
[34] higher enzymatic activities should be obtained in TMOS-gels
than in TEOS-gels. Aging was done only in TrisHCl, since the activity assay mixture would otherwise gelate at pH 7.5. The relative
activities obtained are depicted in Fig. 4.
The relative activities of 36% at pH 6 could be improved by
dilution of the released methanol and the enhancement of the pH
value of the gel until 44%. However it is dazzling that the activity in TMOS-gels was much lower than in TEOS-gels. It suggests
that additional sources for enzyme denaturation must be occurring. Higher shrinkage of TMOS-gels compared to TEOS-gels may
lead to stronger structural changes and reduce enzyme mobility
causing denaturation. Furthermore leakage of enzymes into the
aging buffer solution might cause the loss of activity. Since TMOSgels have pores between 20 and 50 nm individual enzymes might
diffuse into the aging solution.
3.3.3. Aqueous gels
The generation of alcohols during the solgel process can be
avoided by using aqueous routes. Colloidal silica (LUDOX ) was
used as precursor and the aging was performed in TEOS and in
TrisHCl pH 9.5. G6PDH entrapped in aqueous-gel aged in TEOS
conserved 31% of relative activity (Fig. 4). Since these gels contain
many pores smaller than the largest dimension of the enzyme it is
probable that the enzyme is inhibited in its movement. Since aging
at higher pH values leads to larger pore and might lead to more
enzyme mobility [35] aging at pH 9.5 was performed. No activity
was found after aging in TrisHCl pH 9.5. Enzymatic activity losses
by effects of pH might be conceivable. Depending on the buffer and
ionic strength different authors have found highest G6PDH activity
values between 7 and 9 [36,37]. However, the main reason for the
lack of activity may be the complete leaching of the enzyme from
the gel during aging. As can be seen in Fig. 1 the gels had pores
much bigger than the enzyme molecule. It is most likely that the
gels did not contain any enzyme at the moment of performing the
activity measurements.
3.3.4. EGMS-based gels
The relative activity of G6PDH in EGMS-gels after one day of
immobilization in mother liquor (no aging media) was 91%. Aging
in TEOS and TrisHCl (pH 7.5) was also performed (Fig. 4). The difference between the relative activities of the enzyme encapsulated
in EGMS-gels aged in mother liquor and in TEOS is not considerable. The activity of G6PDH immobilized in gels aged in TEOS was
approximately 18% higher than in gels aged in mother liquor. It
can be the result of the protection caused by the polymeric framework which avoids enzyme aggregation and unfolding. The lowest

225

Fig. 5. Carrier specic activities of G6PDH immobilized on Sepabeads EC-HA at

different enzyme loadings (the dashed circle represents the amount of enzyme at
which the carrier capacity is reached).

activity of the samples aged in TrisHCl 7.5 indicates enzymatic

leaching into the aging medium during the aging process.
3.3.5. Covalently immobilized G6PDH on Sepabeads EC-HA
Alternatively to the solgel routes, G6PDH was immobilized
with glutaraldehyde on Sepabeads EC-HA. In this case an average
relative activity of only 12% was reached. The activity of covalently
immobilized enzyme on functionalized surfaces is also strongly
carrier specic, as the less porous Trisoperl amino mediated a signicant higher relative activity of 33%. The particle specic activity
can easily be altered by appropriate choice of enzyme amount during immobilization. According to Fig. 5 the carrier specic activities
showed a linear increase with increasing amount of G6PDH when
the carrier capacity was not exceeded.
3.4. Effect of long-term storage on the biological activity
The effect of long-term storage on the activity of encapsulated
enzymes is an important issue for industrial applications such as
biosensors and biosynthesis. The aging process takes a long time
and might have signicant inuence on the pore structure of the
network and thus on the activity of the encapsulated enzymes.
Long term experiments were performed for G6PDH encapsulated
in TEOS-, TMOS- and EGMS-gels, as well as immobilized with glutaraldehyde on particles. The stability of the encapsulated enzyme
was estimated at 2.5 C in concordance with previous literature
[38,39].
3.4.1. TEOS-based gels
The stability upon storage time of encapsulated G6PDH in TEOSderived gels was investigated within four days of aging in TEOS.
The resulting relative activities are shown in Fig. 6. Activity losses
occurred and a residual activity of 6478% could be preserved. The
half-life of G6PDH in these gels was around 4.5 days. To calculate
the half-life of the encapsulated enzyme a regression with the best
possible correlation was performed with four points (see Fig. 6). The
time to obtain the half of the initial activity value was calculated
(initial activity of G6PDH in TEOS-gels 46.5 Units mg1 ). Activity
losses may be due to several factors: rstly silica may reprecipitate
on the enzyme surface instead of forming a pore around it, in a way
which makes it inaccessible for the substrate. Secondly leaching of
the enzyme into the buffer solution during the aging process could
have taken place. Leaching from gels aged in TEOS is rather improbable due to its immiscibility with water. Another possible cause
of activity losses is the shrinkage occurring during aging, which
reduces the pores and might inhibit the conformational changes
which the enzyme needs to be active. Furthermore the continuing

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S. Cumana et al. / Journal of Molecular Catalysis B: Enzymatic 8586 (2013) 220228

Relative Activity (%)

100

80
60

40
20
0

Fig. 6. Specic activity of free G6PDH () and of G6PDH encapsulated in TEOS- (),
TMOS- () and EGMS-derived () gels depending on the aging time.

release of ethanol during the aging process might be the major reason for the activity losses. It has been observed that although the
encapsulation in TEOS-gels stabilizes the enzymes against ethanol
compared with enzymes in solution, denaturation still occurs. It
may also be due to the increase in ethanol concentration by hydrolysis during aging.
3.4.2. TMOS-based gels
Short-term stability of G6PDH-doped TMOS gels aged in a buffer
solution was tested for 4 days. While the enzymes stored in solution show a constant activity, the entrapped enzymes conserved
only 17% of their activity after four days of aging (see Fig. 6). The
activity of the encapsulated enzyme decreased exponentially and
had a half-life of 3.2 days (initial activity of G6PDH in TMOS-gels
28.2 Units mg1 ). The decrease of activity between the rst and the
fourth day of aging of TEOS and TMOS-gels is comparable. Contrary
to the literature methanol does not seem to be less harmful than
ethanol. A high degree of shrinkage of 27% might have had a greater
negative effect on enzyme structure. Because of the bigger pores in
comparison to TEOS-gels excessive enzyme leaching might have
taken place in TMOS-gels.
3.4.3. EGMS-based gels
The enzymatic activity of G6PDH in EGMS-based gels was investigated for a period of 37 days after encapsulation by aging in
mother liquor. Fig. 6 shows that after this period the residual activity of the enzyme did not sink substantially and was nearly 80%. The
results suggest that EGMS is more compatible with G6PDH than
the other silica precursors used. The activity losses observed during 37 days of aging could be due to residual ethanol generated as
by-product during the preparation of the ethylene glycol modied
silane.
3.4.4. Covalently immobilized G6PDH on Sepabeads EC-HA
Irrespective of the low relative activity, G6PDH immobilized on
Sepabeads EC-HA appeared to be stable over a long period of time
(Fig. 7). Repetitive batch experiments showed that after 154 days
of immobilization the G6PDH kept 77% of its activity directly after
immobilization. Some results found in the literature show that the
time stability of immobilized G6PDH depends strongly on immobilization conditions. For instance Goheer et al. found that the enzyme
retained full activity over a period of 4 months when covalently
attached to CNBr-activated Sepharose 4B; but when attached to
CNBr-activated Sephadex G-25 for 2 weeks lost all of its activity
on storage at room temperature and 40% of its activity on storage at 4 C [40]. Allcock et al. compared G6PDH in solution and
G6PDH immobilized on polyphosphazene/alumina particles [41].
The samples were stored at constant temperature (25 C) and it was
observed that most of the activity of the free enzyme was lost after

50
100
Days after Immobilization

150

Fig. 7. Relative activity of G6PDH on Sepabeads EC-HA during repetitive batch

experiments.

10 days, as a consequence of denaturation of the protein. However

the immobilized enzyme retained a high degree of activity for at
least 90 days.
The long-term experiments of entrapped G6PDH indicate that
alcohol may act in detriment of proteins after encapsulation in
alkoxysilane-based gels. Unfortunately it is not possible to differentiate between the activity losses by denaturation with time and
the losses of activity as product of protein leaching from the gel,
therefore both are considered as a whole. In EGMS-gels the activity
losses are much lower and are comparable with the high G6PDH
stability of the enzyme immobilized via glutaraldehyde on aminofunctionalized particles. The higher compatibility of EGMS with
G6PDH may reside in that it does not generate alcohol during the
solgel reaction.
3.5. Kinetic studies of encapsulated G6PDH in TEOS-based gels
G6PDH
converts
glucose-6-phosphate
(G6P)
to
6phosphoglucono--lactone in the presence of NAD(P)+ .
6-Phosphoglucono--lactone hydrolyzes spontaneously to 6phosphogluconate. The concentration of G6P used in this work
was in the range of 0.023 mM, while keeping the concentration
of NADP+ at the constant level of 0.66 mM. The experiments were
performed with non-crushed TEOS-gels.
The values of turnover number (kcat ) and the specicity constant
(kcat /KM ) for free and encapsulated G6PDH are listed in Table 2. A
comparison of the catalytic constants (kcat ) of both systems reports
that the specic activity of immobilized enzymes is 10% of that of
free G6PDH. Brinker et al. [2] obtained a ratio of 3136% of activity
after encapsulating G6PDH via an aqueous solgel route. The publication afrmed that the immobilization yield was almost 100%
and no leaching occurred. The results published by Brinker et al.
indicate that the enzyme glucose 6-phosphate dehydrogenase is a
labile protein since even an alcohol-free encapsulation route led to
activity losses of 6469%. Considering that the encapsulation route
used in this work leads to the generation of ethanol, and that leaching occurs, the retained activity of 10% is a positive result. In this
work the samples were thicker than in Brinkers work (3 mm vs.
0.8 mm) and hence diffusion paths were longer.
The increase in KM obtained upon encapsulation was 1.3-fold,
which is lower than the results published by Brinker (2.4-fold).
Yamanaka et al. also reported higher increments in the value of
the KM values after encapsulation in TMOS-based gels (approximately 4.5-fold) [42]. The values of kcat in gels are 3.3-fold smaller
than in solution, which represents a slower turnover of substrate by
encapsulated G6PDH. These results can be attributed to diffusion
phenomena occurring within the pores of the gel which difcult
the transport of substrates into and products out of the gel. Some

S. Cumana et al. / Journal of Molecular Catalysis B: Enzymatic 8586 (2013) 220228

227

Table 2
Kinetic parameters for free and encapsulated G6PDH.

Solution
TEOS-gels

KM (mM)

vmax (mol mg1 min1 )

kcat (min1 )

kcat /KM (M1 min1 )

0.181 0.033
0.228 0.051

89.4 3.3
6.2 0.5

1.8E + 8 7E + 6
1.4E + 7 1.2E + 6

9.8E + 5 1.8E + 5
6.1E + 4 8.4E + 3

pores where the enzyme is encapsulated might have also been so

small that the substrates did not have access to it.
3.6. Leaching tests
To determine enzyme leaching in TEOS based gels the Bradford
protein assay was used. The concentration of G6PDH in the samples
was 0.80 0.19 and 0.60 0.13 g ml1 according to the linear plot.
Since the total concentration of the encapsulated G6PDH in the 2 ml
of TrisHCl buffer was 16.6 g ml1 , it could be estimated that 45%
of the encapsulated G6PDH leached into the buffer. Assuming that
the leaching rate of 45% is independent of enzyme concentration,
the major loss of activity found in the experiments must rather
be caused by denaturation during aging of the gels. This is supported by the results from the covalent immobilization of G6PDH
on Sepabeads EC-HA. The enzyme does not seem to substantially
lose activity after 100 days of immobilization. Here, after 5 washing
steps no activity was determined in the supernatants, therefore no
leaching was assumed.
One reason for leaching might be the charge of G6PDH. The isoelectric point of G6PDH is 4.6, which means that at the neutral pH of
the gel the enzyme surface is negatively charged. Since silica is also
negatively charged, there might be electrostatic repulsion between
silica network and enzyme molecules. That enzymes leach out of
the porous network suggests that they do not act as templates for
the formation of the network, or at least not in its whole extension.
To summarize the results suggest that TEOS- and EGMS-based
gels are the best candidates for the immobilization of G6PDH
regarding activity. However time stability regarding leaching and
denaturation is also a selection criterion.
4. Conclusion
This work aimed the encapsulation of G6PDH from L. mesenteroides in silica-based gels from different silica precursors. The
activity of encapsulated G6PDH in TEOS, TMOS, LUDOX and EGMSgels was examined.
Partial evaporation of the alcohol from TEOS-sols did not
improve enzymatic activity, possibly due the stabilizing effect of
the encapsulation. Higher activities were obtained after aging in
TEOS rather than in TrisHCl, and the preservation of almost the
complete activity of the enzyme after one day of encapsulation was
possible. TMOS-gels performed worse than TEOS ones in spite of
generating the less harmful methanol during the gel preparation.
Activity in both alkoxysilane-gels decreased rapidly during 4 days
of encapsulation possibly due to enzyme leaching. This work has
proven that reusability of G6PDH-doped TEOS gels is in principle
possible.
KM values were higher in TEOS-gels than in solution, due possibly to diffusional resistance to transport of substrates in the pores
of the hydrogel. The discrimination of diffusion limitations within
the gels is essentially not feasible. It is mainly due to the difculty
of distinguishing the fraction of the protein which reacts within the
gel and the one which reacts as free enzyme from the solution after
leaching.
G6PDH encapsulated in EGMS-gels preserved almost the whole
initial activity of the enzyme after one day. Minor activity losses
occurred in 37 days due to the absence of solvents and not generation of alcohols during the gel preparation. However residual

alcohol from the preparation of the precursor may lead to the activity losses observed. The activity could even been improved by aging
the gels in TEOS. In comparison G6PDH immobilized on Sepabeads
EC-HA showed lower relative activity than encapsulated enzyme,
but proved to offer high enzymatic stability with time. Encapsulation proved to be a useful alternative to circumvent activity losses
caused by rigidication, non-oriented immobilization, or chemical
modication of residues at the active site or close to it.
Leaching and kinetic measurements are still necessary for further characterization of the gels. Anyway this work demonstrates
that EGMS-gels can be used for the successful encapsulation of
G6PDH and offers a high potential for long term application of
the enzyme. The high compatibility of EGMS with G6PDH suggests
that gels produced from this precursor could be the rst step for
the immobilization of the other enzymes required in the synthetic
pathway for the production of hydrogen.
Acknowledgments
This project was nancially supported by Hamburg Landesexzellensinitiative (Project Synbio) and Joachim Herz Stiftung. The
authors are grateful to Dr. Ins Ardao from the Institute of Bioprocess and Biosystems Engineering of the Technical University of
Hamburg for her valuable contribution to this work.
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