Characterization of cyclodextrin glycosyltransferase immobilized on silica microspheres via aminopropyltrimethoxysilane as a spacer arm

Article abstract of DOI:10.1016/j.molcatb.2012.01.003

In this work, the enzymatic properties of immobilized cyclodextrin glycosyltransferase (CGTase) of Thermoanaerobacter sp. were investigated and compared with the soluble form of the enzyme. CGTase was immobilized on mesoporous silica microspheres synthesized using polyethylene glycol 400 as swelling agent, silanized with 3-aminopropyltrimethoxysilane (APTMS), and activated with glutaraldehyde prior to immobilization. This innovative approach for support preparation produced high yields of immobilization (83%) and activity recoveries (73%), which are the highest so far reported for CGTase. The soluble enzyme (CGTase) and its immobilized form (ImCGTase) showed similar values for the optimal pH activity, while optimal reaction temperatures were found to be 100 °C and 80 °C, respectively. The immobilized enzyme showed similar values for K_m and thermal stabilities with the soluble form, while its V_max was lower. The immobilized enzyme was tested in repeated batches in order to simulate recovery and reuse, keeping about 60% of the initial catalytic activity after 15 cycles, showing its good chemical and mechanical resistance.

Full text of DOI:10.1016/j.molcatb.2012.01.003

Journal of Molecular Catalysis B: Enzymatic 78 (2012) 51–56
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Characterization of cyclodextrin glycosyltransferase immobilized on silica
microspheres via aminopropyltrimethoxysilane as a “spacer arm”
Carla Roberta Mattea, Michael Ramos Nunesb, Edilson Valmir Benvenuttib,
Jéssie da Natividade Schöffera, Marco Antônio Záchia Ayuba, Plinho Francisco Hertza,∗
a Food Science & Technology Institute (ICTA), Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
b Chemistry Institute (IQ), Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, the enzymatic properties of immobilized cyclodextrin glycosyltransferase (CGTase) of Ther-
moanaerobacter sp. were investigated and compared with the soluble form of the enzyme. CGTase was
immobilized on mesoporous silica microspheres synthesized using polyethylene glycol 400 as swelling
agent, silanized with 3-aminopropyltrimethoxysilane (APTMS), and activated with glutaraldehyde prior
to immobilization. This innovative approach for support preparation produced high yields of immo-
bilization (83%) and activity recoveries (73%), which are the highest so far reported for CGTase. The
soluble enzyme (CGTase) and its immobilized form (ImCGTase) showed similar values for the optimal
pH activity, while optimal reaction temperatures were found to be 100 ◦C and 80 ◦C, respectively. The
immobilized enzyme showed similar values for Km and thermal stabilities with the soluble form, while
its Vmax was lower. The immobilized enzyme was tested in repeated batches in order to simulate recovery
and reuse, keeping about 60% of the initial catalytic activity after 15 cycles, showing its good chemical
and mechanical resistance.
Received 15 October 2011
Received in revised form
21 December 2011
Accepted 6 January 2012
Available online 16 February 2012
Keywords:
Cyclodextrin glycosyltransferase
Enzyme immobilization
Toruzyme®
Thermoanaerobacter sp.
Silica
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
overcome these problems, the addition of spacers, or linkers, has
been investigated to produce more flexible spatial arrangements
Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is a
microbial enzyme capable to convert starch and related polymers
into cyclodextrins (CDs) via cyclization reactions. Cyclodextrins
have the ability of encapsulating hydrophobic molecules into their
internal cavity, promoting the stabilization, solubilization, and
the masking of odors and tastes of a wide variety of important
compounds that are used in foods, pharmaceuticals, cosmetics,
agricultural products and chemicals [1–10].
The industrial production of CDs has been chiefly carried out
in batch systems, with soluble CGTase used directly into the reac-
tion mixture. The use of immobilized CGTase has been proposed
as a technical alternative to allow for its continuous and repeated
use, avoiding enzyme solubilization and improving the stability
of the biocatalyst [11]. However, it has been reported that activ-
ity recoveries of immobilized CGTases are usually very low due
to diffusional resistances imposed by the structure of the macro-
molecular substrates, such as starches and dextrins [11–13]. To
of the biomolecules, providing higher mobility of the resulting
immobilized enzyme, while enhancing the interaction between the
enzyme and the macromolecular substrate [14,15].
A wide variety of materials have been used as immobilization
supports for CGTase such as Eupergit C [7], commercial chitosan
[16,17], glyoxyl-agarose [18], controlled pore size silica [16,19],
among others. In particular, silica supports have been shown to be
excellent materials for enzyme immobilization due to their excel-
lent biocompatibility, rigidity, mechanical and operational stability,
high thermal resistance, and nontoxicity [20–22]. It also shows
remarkable resistance to microbial degradation and to solvent dis-
tortion [23]. Surface modification via functional groups provides
the opportunity to change the interfacial properties, while retaining
their basic geometry and mechanical strength of the solid matrix
in cyclic reuse [21].
Several enzymes have been immobilized in a variety of silica
matrices using different methods. In this study, it is demonstrated
the use of an emulsion for preparing ordered silica microspheres
with the PEG polymer as the swelling agent [24]. Chemical attach-
ment of Thermoanaerobacter sp. CGTase by covalent coupling onto
silica microspheres silanized with 3-aminopropyltrimethoxysilane
(APTMS) was the chosen mechanism because it can generate a
highly stable system [25–27]. In this system, a solution of APTMS
∗
Corresponding author at: Food Science & Technology Institute (ICTA), Federal
University of Rio Grande do Sul (UFRGS), Av. Bento Gonc¸ alves, 9500, PO Box 15095,
ZC 91501-970, Porto Alegre, RS, Brazil. Tel.: +55 51 33087094; fax: +55 51 33087048.
E-mail address: plinho@ufrgs.br (P.F. Hertz).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2012.01.003

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C.R. Matte et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 51–56
Fig. 1. Schematic diagram of the process for the preparation of CGTase immobilization on silica.
reacts with hydroxyl groups or silanol supports (the “silanization”
process), introducing a spacer arm between the enzyme molecule
and the support. This arrangement facilitates the access of the
immobilized enzyme to its reactional substrate [28]. The effects
of this immobilization process on pH reaction and temperature,
the kinetic parameters, and the stability of ImCGTase were also
investigated and compared with the soluble form of the enzyme.
2.3. Synthesis and functionalization of silica–polyethylene glycol
microspheres
The silica microspheres were prepared according to Yang et al.
[22] with the following modifications. One aliquot of 5 mL of
tetraethylorthosilicate (TEOS) was mixed with water at a molar
ratio of 0.015:1 and acidified with HCl; the pre-hydrolysis of
TEOS was then obtained by adding 0.5 mL polyethylene glycol
400 (PEG-400) to this mixture under vigorous stirring. When the
emulsification was complete, 0.5 mL of the dicyclohexylamine cat-
alyst was added in order to start the polycondensation reactions.
The microspheres were produced in the form of hydrogel, which
resulted in solid spheres by dehydration. After aging and drying for
15 days at room temperature, the hydrogel beads were successively
washed with water and ethanol.
The silica microspheres were functionalized with the addition of
1 mL of aminopropyltrimethoxysilane (APTMS)/g of microspheres
under reflux in toluene for 24 h. The resulting product was washed
several times with toluene, ethanol, ether, and water, and finally
dried under vacuum at 60 ◦C for 4 h.
2. Materials and methods
2.1. Enzyme and chemicals
CGTase from Thermoanaerobacter sp. (Toruzyme 0.2L) was
kindly provided by Novozymes A/S (Bagsvaerd, Denmark). ␤-CD
was purchased from Sigma–Aldrich (St. Louis, USA). All other chem-
icals were of analytical grade and purchased either from Fluka or
Merck (São Paulo, Brazil).
2.2. Assay of ˇ-CD forming activity
The pore size distribution was estimated using the BJH method
[31] and the specific surface area was determined by the BET mul-
tipoint technique [32] using adsorption of N2 at −196 ◦C. Images
of the support were taken by scanning electron microscopy (SEM;
JEOL, model JSM 6060, Japan).
CGTase cyclization activity was determined using the phenolph-
thalein assay [29] with modifications as described by Pinto et al.
[30], based on the ability of the formed cyclodextrin to form
inclusion complexes with phenolphthalein. The reaction mixture,
containing 26 mg of soluble starch in 0.65 mL of appropriate buffer,
and 0.35 mL of commercial enzyme, was incubated at appropriated
temperature for 15 min. The reaction was stopped with the addition
of 4 mL of 0.04 mM phenolphthalein dissolved in 125 mM Na2CO3
solution. The absorbance was then measured at 550 nm. One unit
of CGTase activity was defined as the amount of enzyme to produce
1 ␮mol of ␤-CD/min. The measurement of immobilized CGTase
activity (ImCGTase) was performed using the reaction supernatant
after centrifugation at 3500 × g for 15 min at 4 ◦C.
2.4. Immobilization on silica
Functionalized silica (0.5 g) was immersed in a solution of 0.5 mL
of glutaraldehyde (volume fraction 25%) and 4.5 mL of 20 mM
sodium phosphate buffer (pH 7.0); the mixture was stirred for 1 h at
room temperature. The activated support was extensively washed
with the same buffer and immediately incubated under agitation
in an orbital shaker (200 rpm), added of 10 mL of CGTase solution

C.R. Matte et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 51–56
53
Fig. 2. CGTase relative activity as a function of pH (buffers 0.1 M sodium acetate for
pH 4 and 5; 0.1 M sodium phosphate for pH 6, 7, and 8; and 0.1 M glycine–NaOH
for pH 9 and 10) at 60 ◦C. (᭹) CGTase, soluble enzyme. (ꢀ) ImCGTase, immobilized.
Results are the mean of triplicates.
(approximately 200 U/mL in 0.1 M sodium phosphate buffer, pH
6.0) at 8 ◦C for 15 h. The schematic representation of the immo-
bilization process is shown in Fig. 1.
After immobilization, successive washings with buffer were
used to remove the excess of enzyme until no activity could be
detected in the washed fractions. The immobilized enzyme sys-
tem was also washed with 1 M NaCl and ethylene glycol (volume
fraction 30%) in order to eliminate any enzyme molecule non-
covalently attached to the support.
2.5. Effect of pH and temperature on the CGTase activity
The effects of immobilization on the enzyme activities were
measured for the range of pH varying from 4.0 to 10.0 at 60 ◦C,
using either sodium acetate buffer 0.1 M (pH 4 and 5), sodium phos-
phate buffer 0.1 M (pH 6, 7 and 8), or glycine–NaOH buffer (pH 9
and 10). The effects of temperature on CGTase activities were mea-
sured in the presence of sodium phosphate buffer 0.1 M, pH 6.0,
in the range varying from 50 to 120 ◦C. Reactions were carried out
using the CGTase assay procedure described in Section 2.2.
Fig. 4. Lineweaver–Burk plots of CGTase, soluble enzyme (A) and ImCGTase, immo-
bilized (B). Results are the mean of triplicates.
2.6. Kinetic parameters of the soluble and immobilized CGTases
The kinetic parameters were determined by incubating the
diluted enzyme (approximately 200 U/mL) with 2.5–60 mg/mL
Fig. 3. CGTase activity as a function of temperature (50–120 ◦C) using 0.1 M sodium
phosphate buffer pH 6. (᭹) CGTase, soluble enzyme. (ꢀ) ImCGTase, immobilized.
Results are the mean of triplicates.
Fig. 5. Storage stability in fridge at 8 ◦C for 30 days. (᭹) CGTase, soluble enzyme. (ꢀ)
ImCGTase, immobilized. Results are the mean of duplicates.

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C.R. Matte et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 51–56
Fig. 7. Relative activities for the cyclization of starch with multiple uses. Cyclization
reaction was performed in sodium phosphate buffer solution (0.1 M, pH 6) for 15 min
at 80 ◦C and 200 rpm rotation. Results are the mean of triplicates.
Fig. 6. Thermal stability at different temperatures. (᭹) CGTase at 60 ◦C. (ꢁ) ImCG-
Tase at 60 ◦C. (ꢀ) CGTase at 80 ◦C. (ꢁ) ImCGTase at 80 ◦C. (ꢂ) CGTase at 100 ◦C. (ꢀ)
ImCGTase at 100 ◦C. Results are the mean of duplicates.
recoveries of activities obtained in this work were approximately
seven and two-fold higher, respectively [7,18], being the highest
ever reported for ImCGTase.
solutions of soluble starch in 0.1 M sodium phosphate pH 6 at the
optimal temperature for 15 min. The values of Km and Vmax were
then determined using the Lineweaver–Burk plot.
3.2. Effects of pH and temperature on the activities of CGTase and
ImCGTase
2.7. Storage and thermal stability of immobilized CGTase
Immobilized and soluble enzymes were stored in the fridge at
8 ◦C for 30 days. The remaining enzyme activity was measured
with phenolphthalein at optimal pH and temperature for 15 min
as described above. Thermal inactivation assays were performed at
60, 80, and 100 ◦C in 0.1 M sodium phosphate pH 6. Aliquots were
periodically taken and the activity was measured as described in
Section 2.2.
Fig. 2 presents the results for the enzyme activities as a function
of pH. Results show that at 60 ◦C, the optimal pH was 6.0, with no
differences found for soluble and immobilized CGTases. The activi-
ties of soluble CGTases were kept elevated in acid pH, as it has been
reported by other authors [11].
The optimal temperatures for CGTase and ImCGTase were eval-
uated in the range varying from 50 to 120 ◦C at the optimal pH 6
for the catalytic activity. The highest activity for the soluble enzyme
was achieved at 100 ◦C, suggesting its excellent thermostability. On
the other hand, the ImCGTase presented best activities between
70 and 90 ◦C, remaining constant in this range, as shown in Fig. 3.
These high reactional temperatures are very important for the pos-
sible industrial applications of ImCGTase. Zhou et al. [33] showed
that the optimal temperature for Toruzyme of Thermoanaerobac-
ter sp. was 100 ◦C, while Unsworth et al. [34], reported that several
thermophilic enzymes, such as ␣-amylases from Pyrococcus furiosus
and Methanococcus jannaschii, have optimal reaction temperatures
between 106 and 120 ◦C, respectively.
2.8. Operational stability of the immobilized CGTase
For the reuse tests of the ImCGTase, the immobilized enzyme
system was incubated under the optimal conditions (80 ◦C, pH
6) for CD production. At the end of each cycle, the immobilized
enzyme was separated from the reactional product by centrifuga-
tion at 3500 × g for 15 min at 4 ◦C and the activity was measured
in the supernatant. The ImCGTase was thoroughly washed with
0.1 M sodium phosphate (pH 6) and finally resuspended in a freshly
prepared substrate solution to restart a new cycle.
Conformational changes in the enzyme molecule could explain
this change in the best temperature for optimal activity when sol-
uble and immobilized CGTase are compared. Small decreases in
the optimal temperature have also been reported for Thermoanaer-
obacter sp. ImCGTase by other authors [7]. Nevertheless, at this
range of temperatures, ImCGTase could be used in industrial pro-
cesses ensuring that no microbial contamination would take place.
Moreover, at 80 ◦C, the solubility of substrates and products are
high, possibly increasing productivity and yields, while reducing
the costs of industrial applications.
3. Results and discussion
3.1. Immobilization of CGTase on silica microspheres and support
characterization
Analysis of silica microspheres showed that the specific sur-
face area of the support was 185.04 m2/g and the pore volume
was 0.08 cm3/g, with mesoporous structure (pore diameter 4.6 nm)
being in accordance with the standards recommended by the Inter-
national Union of Pure and Applied Chemistry.
The activated surface of these mesoporous microspheres was
effective for enzyme immobilization. It can be observed that the
yield of immobilization reached 83% and the recovery of activity
was 73%. Table 1 shows the measured parameters for immobi-
lization of CGTase on silica microspheres support. These results
clearly show the high efficiency of silica microspheres immobiliza-
tion system for the CGTase of Thermoanaerobacter sp. Compared
with eupergit C and glyoxyl-agarose immobilized systems, the
3.3. Kinetic parameters
The kinetic parameters calculated using the Lineweaver–Burk
plots are listed in Table 1 and shown in Fig. 4. The lower Vmax
values for the immobilized enzyme might be due to the conforma-
tional changes caused by the fixation of the enzyme to the support.
Alternatively, it might reflect the constraints for the substrate to
access the active site of the enzyme [35]. Similar changes in Vmax

C.R. Matte et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 51–56
55
Table 1
Parameters obtained for the CGTase immobilized onto silica microspheres compared with the soluble enzyme.
Offered activity
(U/mL)
Non-linked
activity (U/mL)
Immobilized
yield (%)
Immobilized
enzyme activity
(U/mL)
Activity
recovery (%)
Km (mg/mL)
Vmax (U/mL)
ImCGTase
CGTase
192
–
32
–
83
–
117
–
73
–
27
27
125
167
Fig. 8. Scanning electron micrograph (SEM). (A) Silica microspheres before activation with glutaraldehyde (10 kV; 500×); (B) silica microspheres after 15 batches (20 kV;
500×).
were observed for other immobilization systems using PVC [12]
and alumina [35] as supports, with CGTases of different microbial
sources.
The physical stability of the support is an important factor
because it prevents the loss of the enzyme to the reaction medium,
which would contaminate the product, making more difficult its
purification [36]. In Fig. 8 is presented the scanning electron micro-
graph (SEM) of the structure of the silica microspheres after 15
cycles. The pictures clearly show the exceptionally good mechan-
ical resistance of the system, with no structural modifications
appearing in the microphotographies.
3.4. Storage and thermal stabilities
The storage and thermal stabilities of immobilized enzymes are
important parameters concerning their possible industrial applica-
tions [35]. Fig. 5 shows the results for the storage stabilities of the
soluble and immobilized CGTases. The activity of ImCGTase was
practically unchanged for the first 18 days of storage. However,
after 30 days in the refrigerator, the remaining ImCGTase activity
was 60%, lower than that of the soluble enzyme, which was 85%
after this time.
Concerning the thermal stability, shown in Fig. 6, the ImCG-
Tase presented practically the same characteristics of the soluble
enzyme at 60 and 80 ◦C, with activities kept above 90% after 5 h
of incubation. However, at 100 ◦C a fast decrease was observed,
with 85% of activity loss in 0.5 h for both free and immobilized
enzymes. Nevertheless, the immobilized system used in this work
performs better than other immobilized CGTases. For instance,
Amud et al. [11] reported that the sol-gel-encapsulated CGTase
from Thermoanaerobacter sp., incubated at 60 ◦C and pH 6.0 (citrate
buffer), was almost completely inactivated after 3 h of reaction. The
same enzyme, when immobilized on Eupergit C, showed good ther-
mostabilities at temperatures of 60 and 85 ◦C, but retained only 40%
of its relative activity after 1 h of incubation at 95 ◦C [7].
4. Conclusions
There are many important reasons for using immobilized
enzymes, which includes the convenience of enzyme preparation,
handling, and downstream separation of enzyme and its reactional
products. Moreover, the possibility of enzyme reuse in successive
batches, or in a continuous enzyme reactor, is of foremost impor-
tance. In particular, enzyme reuse provides cost advantages, which
are often an essential prerequisite in order to establish an econom-
ically viable enzyme catalysis process [37,38]. In this work, it was
possible to successfully immobilize Thermoanaerobacter sp. CGTase
using a new approach on silica microspheres, showing high immo-
bilization yields and activities recoveries, which could be used up to
11 batches reaction cycles almost without any decrease in its rela-
tive activity. The results obtained here show better performances of
ImCGTase compared with other immobilization systems reported
in the literature. The operational stability of Thermoanaerobacter sp.
ImCGTase on silica microspheres presented in this work points out
to its possible applicability in the industrial production of cyclodex-
trins, granting further research on the scaling-up of this enzymatic
process.
3.5. Stability of ImCGTase over repeated batches processes
It is highly desirable from the industrial point of view that an
enzymatic system could be reused several times without losing its
activity, thus reducing process costs. The stability of the immobi-
lized CGTase over repeated batches was evaluated in a 15 cycles
process and the results are shown in Fig. 7. It was found that the
immobilized enzyme produced a satisfactory yield of ␤-CDs after
successive runs, retaining 60% of the initial catalytic activity at the
end of the 15 cycles. Up to the 11th cycle, activity losses were not
detected. Again, the silica microspheres system performed well
compared with other systems. For instance, the operational sta-
bility of immobilized Thermoanaerobacter sp. CGTase on Eupergit C
reported by Martín et al. [7] showed that after 8 cycles the system
kept only 40% of residual activity.
Acknowledgments
The authors wish to thank Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) and Coordenac¸ ão de Aperfoic¸ oamento
de Pessoal de Nível Superior (CAPES), both Brazilian bureaus for sci-
ence and education, for their financial support and scholarships for
this project.
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