Effects of immobilization, pH and reaction time in the modulation of α-, β- or γ-cyclodextrins production by cyclodextrin glycosyltransferase: Batch and continuous process

Carbohydrate Polymers 169 (2017) 41–49

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Effects of immobilization, pH and reaction time in the modulation of
˛-, ˇ- or -cyclodextrins production by cyclodextrin
glycosyltransferase: Batch and continuous process
Jéssie da Natividade Schöffer a , Carla Roberta Matte a , Douglas Santana Charqueiro b ,
Eliana Weber de Menezes b , Tania Maria Haas Costa b , Edilson Valmir Benvenutti b ,
Rafael C. Rodrigues a , Plinho Francisco Hertz a,∗,1
a
Grupo de Biotecnologia, Bioprocessos e Biocatálise, Instituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul (UFRGS),
Porto Alegre, RS, Brazil
b
Laboratório de Sólidos e Superfície, Instituto de Química, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil

a r t i c l e

i n f o

Article history:
Received 6 February 2017
Received in revised form 28 March 2017
Accepted 3 April 2017
Available online 4 April 2017
Keywords:
CGTase immobilization
CDs modulated production

Packed-bed reactor
CDs continuous production

a b s t r a c t
This study reports the immobilization of a ˇ-CGTase on glutaraldehyde pre-activated silica and its use to
production of cyclodextrins in batch and continuous reactions. We were able to modulate the cyclodextrin
production (˛-, ˇ- and -CD) by immobilization and changing the reaction conditions. In batch reactions,
the immobilized enzyme reached to maximum productions of 4.9 mg mL−1 of ␣-CD, 3.6 mg mL−1 of ˇ-CD
and 3.5 mg mL−1 of -CD at different conditions of temperature, pH and reaction time. In continuous
reactor, varying the residence time and pH it was possible to produce at pH 4.0 and 141 min of residence
time preferentially -CD (0.75 and 3.36 mg mL−1 of ␣- and -CD, respectively), or at pH 8.0 and 4.81 min
␣- and ˇ-CDs (3.44 and 3.51 mg mL−1 ).
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Cyclodextrins (CDs) are cyclic oligosaccharides containing
mainly six (␣-CD), seven (ˇ-CD) or eight ( -CD) glucose residues,
linked by ␣(1-4) glycosidic bonds. Due to the conformation of
their glucose residues and the links established among them, CDs
have an unique spatial configuration, showing a cylindrical hollow
truncated cone shape with a hydrophobic nanoscale cavity and a
hydrophilic outer surface (Loftsson & Duchene, 2007; Szejtli, 1982,
1998).
The most notable feature of CDs is their ability to form inclusion complexes with solid, liquid and gaseous molecules that fit
into their hydrophobic cavity (Del Valle, 2004; Singh et al., 2002).
The CDs architecture provides a wide range of applications in phar-

∗ Corresponding author at: Instituto de Ciência e Tecnologia de Alimentos (ICTA),
Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonc¸alves, 9500, P.O.
Box: 15095, ZC 91501-970, Porto Alegre, RS, Brazil.

E-mail addresses: (R.C. Rodrigues),
(P.F. Hertz).
1
Web: /> />0144-8617/© 2017 Elsevier Ltd. All rights reserved.

maceuticals (Lima et al., 2016; Moussa, Hmadeh, Abiad, Dib, &
Patra, 2016; Pereva, Sarafska, Bogdanova, & Spassov, 2016), food
(Astray, Gonzalez-Barreiro, Mejuto, Rial-Otero, & Simal-Gandara,
2009; Astray, Mejuto, Morales, Rial-Otero, & Simal-Gandara, 2010;
Li, Chen, & Li, 2017; Yuan, Du, Zhang, Jin, & Liu, 2016; Zhao & Tang,
2016), cosmetic and textile processing industries (Mihailiasa et al.,
2016). As described by Singh et al. (2002), CDs are multipurpose
technological tools, they can stabilize and protect the encapsulated
molecules from volatility and oxidation, enhance their apparent
solubility, hydrophilicity and bioavailability, reduce adverse effect
of pharmaceuticals and protect the substances against any undesirable reactions (Astray et al., 2009; Del Valle, 2004; Singh et al.,
2002).
CDs are produced from starch as a result of intramolecular transglycosylation, one of the four reactions catalyzed by the enzyme
cyclodextrin glycosyltransferase (CGTase). The specificity and the
yield of each CD depends on the enzyme, resulting in a mixture
of linear, branched and cyclic dextrins (␣-, ˇ-, and -CD) at different concentrations. Furthermore, for most of the enzymes, the
main products are ␣- and ˇ-CDs, and few CGTases produce -CD as
the main product (Kamaruddin, Illias, Aziz, Said, & Hassan, 2005;
Li et al., 2007). Because the specificity of the molecular inclusion


42

J.d.N. Schöffer et al. / Carbohydrate Polymers 169 (2017) 41–49

Table 1
Experimental designs and results of the CCD.
Experiments

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

Variables

Results

X1 (temperature, ◦ C)

X2 (pH)

X3 (time, h)

˛-CD (mg mL−1 )

ˇ-CD (mg mL−1 )

−1 (58.1)
−1 (58.1)
−1 (58.1)
−1 (58.1)
+1 (81.9)
+1 (81.9)
+1 (81.9)
+1 (81.9)
−1.68 (50)
+1.68 (90)
0 (70)
0 (70)
0 (70)
0 (70)
0 (70)
0 (70)
0 (70)
0 (70)

−1 (4.8)
−1 (4.8)
+1 (7.2)
+1 (7.2)

−1 (4.8)
−1 (4.8)
+1 (7.2)
+1 (7.2)
0 (6)
0 (6)
−1.68 (4)
+1.68 (8)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)

−1 (9.9)
+1 (38.3)
−1 (9.9)
+1 (38.3)
−1 (9.9)
+1 (38.3)
−1 (9.9)
+1 (38.3)
0 (24.1)
0 (24.1)
0 (24.1)
0 (24.1)
−1.68 (0.25)
+1.68 (48)
0 (24.1)

0 (24.1)
0 (24.1)
0 (24.1)

2.29
1.07
3.28
1.42
2.70
1.04
3.16
1.25
1.87
4.93
1.36
2.81
2.45
0.72
1.27
1.37
1.20
1.29

2.24
0.39
3.63
1.18
2.11
0.32
2.88

0.67
1.30
3.09
0.42
2.92
1.34
0.14
0.87
0.92
0.78
0.91

Fig. 1. Thermogravimetric analyses of silica and its derivatives with different
amounts of amino groups. (solid black line) Si, (dashed line) Si-NH-0.29, (dotted
line) Si-NH-0.45, (dash-dot line) Si-NH-0.65.

process, as well as the different properties and possibilities of application of each CD (Lima et al., 2016; Szente et al., 2016), the target
product will define the choice of the appropriate enzyme by the
industry. On contrary, exhaustive crystallization steps or the use
of solvents will be necessary to obtain and purify a particular CD
(Blackwood & Bucke, 2000; Ferrarotti et al., 2006; Li, Chen, Gu, Chen,
& Wu, 2014; Li et al., 2007; Szejtli, 1998). In this sense, changes in
yield of the three main CDs by varying the sources of the enzyme
and substrate have been studied by several authors. Currently, the
focus is the use of genetic mutations in order to direct the production of specific CDs (van der Veen, Uitdehaag, Dijkstra, & Dijkhuizen,
2000; van der Veen, Uitdehaag, Penninga et al., 2000; Xie, Song, Yue,
Chao, & Qian, 2014; Yamamoto et al., 2000).
Since CDs are produced exclusively by enzymatic means, techniques such as enzyme immobilization are widely assessed in
order to improve the enzyme stability and achieve greater control of the reaction (Graebin et al., 2016). Supports such as silica,
chitosan, polyethylene films and agarose are among the most

used for immobilization of this enzyme using different approaches
such as adsorption, entrapment and covalent binding (Matte
et al., 2012; Schöffer, Klein, Rodrigues, & Hertz, 2013; Sobral
et al., 2003; Sobral, Rodrigues, de Oliveira, de Moraes, & Zanin,
2002). Immobilized CGTases generally exhibit greater resistance
to changes in conformations caused by denaturing conditions,

-CD (mg mL−1 )
0.89
2.41
0.81
1.63
1.26
2.71
1.02
2.53
1.56
1.44
3.52
2.01
0.38
2.75
1.93
2.17
2.01
2.01

making them more resistant to variations of pH and temperature (Abdel-Naby, 1999; Martín, Plou, Alcalde, & Ballesteros, 2003;
Tardioli, Zanin, & de Moraes, 2006). Moreover, the immobilization
allows the use of the biocatalyst in continuous reactors, achieving higher productivity and lower production cost (Garcia-Galan,

Berenguer-Murcia, Fernandez-Lafuente, & Rodrigues, 2011; Mateo,
Palomo, Fernandez-Lorente, Guisan, & Fernandez-Lafuente, 2007;
Rodrigues, Ortiz, Berenguer-Murcia, Torres, & Fernandez-Lafuente,
2013).
Thus, the objective of the present study was to evaluate the
effects of reaction conditions in the specific production of ␣-, ˇ- and
-CD by an immobilized ˇ-CGTase. The enzyme from Thermoanaer®
obacter sp. (Toruzyme 3.0 L ) was immobilized in mesoporous
silica functionalized with 3-aminopropyltrimethoxysilane and
activated with glutaraldehyde, that provide a covalent reaction by
the most reactive amino group on the enzyme surface (at neutral pH
value, the reaction occurs with the terminal amino group) (Barbosa
et al., 2014). The immobilized preparation was applied in batch
and continuous reactions varying the temperature, pH and time
using a central composite design (CCD) and the response surface
methodology (RSM), so it was possible to modulate the production
of specific CDs according to reaction conditions.
2. Experimental section
2.1. Materials
®

Thermoanaerobacter sp. CGTase (Toruzyme 3.0 L) was kindly
provided by Novozymes A/S (Bagsvaerd, Denmark). Tetraethylorthosilicate 98% (TEOS), 3-aminopropyltrimethoxysilane 97%
(APTMS), ␣- ˇ- and -CD were obtained from Sigma-Aldrich (St.
Louis, USA). All other reagents used were of analytical grade.
2.2. Synthesis and functionalization of mesoporous silica
The mesoporous silica support for enzyme immobilization was
synthesized by sol-gel method. A mixture containing 5 mL of TEOS,
5 mL of ethanol and catalyst were stirred. The catalyst consists of
36 drops of HF/HCl (6/6 mol L−1 ) mixture in 2 mL of water solution.

This mixture was stored during 2 weeks at ambient temperature
for gelation. Then, the formed xerogel was comminuted, washed
with water and ethanol, and dried for 2 h at 90 ◦ C, under vacuum
(Caldas et al., 2017). The silica support was functionalized with
APTMS providing amino groups on its surface. The organofunction-


J.d.N. Schöffer et al. / Carbohydrate Polymers 169 (2017) 41–49

Fig. 2. Cyclodextrins production by (a) free CGTase, 50 ◦ C; (b) free CGTase, 70 ◦ C; (c) immobilized CGTase, 50 ◦ C; (d) immobilized CGTase, 70 ◦ C. (᭿) ␣-CD, (
␥-CD.

alization was made using 0.25, 0.5 and 1 mmol of APTMS precursor
per gram of silica. The reaction was performed in toluene at 80 ◦ C, in
argon atmosphere, under mechanical stirring, for 24 h. Afterwards,
the supernatant was removed and the silica supports were washed
with toluene, ethanol, water, and dried in vacuum at 80 ◦ C, for 2 h.
Further, the samples of silica functionalized with amino groups
were activated with glutaraldehyde to make them able to bind the
enzyme via amino-terminal. Then, 0.5 mL of glutaraldehyde solution (5% v/v at pH 7.0) was mixed to 10 mg of support during 2 h
and then, washed several times to remove the excess of activation
agent.
Before and after functionalization, activation and immobilization, the materials were lyophilized and submitted to N2
adsorption-desorption and thermogravimetric analysis (TGA) for
textural characterization and to ensure the binding of the organic
groups.
The thermogravimetric analysis was performed on a Shimadzu
Instrument model TGA-50H, under argon flow at 50 mL min−1 ,
with a heating rate of 10 ◦ C min−1 , from room temperature up to
650 ◦ C. N2 isotherms were obtained at liquid nitrogen boiling point

using a Tristar 3020 Kr Micromeritics equipment. Samples were
previously degassed at 120 ◦ C, under vacuum, for 12 h. The specific

43

) ␤-CD, (

)

surface areas were estimated by the Brunauer, Emmett and Teller
(BET) multipoint method and the pore size distributions were calculated by using Barret, Joyner and Halenda (BJH) model applied to
the desorption branch of isotherms (Gregg & Sing, 1982).
2.3. Enzyme immobilization
For enzyme immobilization, 0.5 mL of CGTase solution (in
sodium phosphate buffer 10 mmol L−1 , pH 6.0) and 10 mg of activated support (protein load of 10 mg g−1 ) were mixed overnight
under gentle stirring. After that, the support was separated by
decantation from the solution and washed with sodium phosphate buffer (10 mmol L−1 , pH 6.0), ethylene glycol (30%) and NaCl
(1 mol L−1 ) to remove non-covalently bonded proteins.
Enzyme concentration of immobilization solution and washing fractions were determined by enzymatic activity assay. These
values were used to calculate the immobilization yield and efficiency, using the equations below, according to Sheldon and van
Pelt (2013). The protein concentrations were determined according to Lowry method (Lowry, Rosebrough, Farr, & Randall, 1951).
Yield (%) = 100 × (immobilized activity/starting activity)


44

J.d.N. Schöffer et al. / Carbohydrate Polymers 169 (2017) 41–49

Table 2
Textural and thermal analyses data.

Sample

Specific surface area
(±7 m2 g−1 )

Pore volume
±0.01 cm3 g−1 )

Pore diametera (nm)

Amino groupb (mmol g−1 )

Si
Si-NH-0.29
Si-NH-0.45
Si-NH-0.65

133
121
114
109

0.79
0.72
0.65
0.61

24
23
23

21

–
0.29
0.45
0.65

a
b

Maximum of BJH pore diameter distribution curve.
Aminopropyl group amount estimated by TGA.

Table 3
CGTase immobilization parameters on Si-NH-G.

2.7. Central composite design

Sample

Yield (%)

Efficiency (%)

U g−1 a

Si-NH-0.29-G
Si-NH-0.45-G
Si-NH-0.65-G

96.54
98.18
98.63

3.41
3.92
5.37

6333
7405
10173

a

Measured by the phenolphthalein method.

Efficiency (%) 100 × = (observed activity/immobilized activity)

2.4. Enzymatic activity
The CGTase activity was determined by using the phenolphthalein method developed by Vikmon (1982), with some
modifications. This method is based on the decrease in the color
of a phenolphthalein solution caused by its encapsulation by ˇCD formed during the reaction. The substrate solution was soluble
starch 4% (w/v) in sodium phosphate buffer 10 mmol L−1 , pH 6.0.
For free CGTase activity, 1.05 mL of enzyme solution and 1.95 mL of
substrate were mixed and incubated at 60 ◦ C for 15 min. For immobilized CGTase, 1.95 mL of substrate solution was added to 1.05 mL
of sodium phosphate buffer containing 10 mg of immobilized
enzyme and incubated at 60 ◦ C for 10 min, under gentle stirring.
At the end of the reaction time, 0.5 mL of this mixture was added to
2 mL of phenolphthalein solution (0.04 mmol L−1 phenolphthalein
dissolved in 125 mmol L−1 Na2 CO3 ). The decrease in color intensity

was measured in a spectrophotometer at 550 nm, and the ˇ-CD
concentration was determined by applying the absorbance value
at a standard curve with the range of 40–400 ␮g mL−1 of a commercial ˇ-CD. One unit of CGTase activity (U) was defined as the
amount of enzyme that produces 1 ␮g ˇ-CD min−1 under the reaction conditions.
2.5. Cyclodextrins quantification
Quantifications of ␣-, ˇ- and -CDs were determined using a
HPLC system (Shimadzu, Tokyo, Japan) equipped with a refractive
index detector (Shimadzu, RID-10A) and Aminex HPX-42A column
(Bio-Rad). Distilled water was used as mobile phase at 70 ◦ C and
a flow rate of 0.5 mL min−1 . The samples and mobile phase were
filtered through Millipore membranes of 0.22 ␮m.
2.6. Production of cyclodextrins as a function of time
Preliminary tests were carried out for cyclodextrins production under different conditions of pH, temperature and reaction
time using free (95.82 U) and immobilized enzyme (101.73 U). The
temperatures evaluated were 50 and 70 ◦ C. The substrate was soluble starch 4% (w/v) at pH 4.0 and 7.0 (sodium phosphate buffer,
10 mmol L−1 ). Samples were taken at 1, 24 and 48 h, filtered and
analyzed by HPLC for CDs quantification.

A 23 central composite design (CCD) with three variables was
performed to evaluate the combined effects of temperature, pH and
reaction time in the production of ␣-, ˇ- and -CD by immobilized
CGTase (10 mg). The temperature varied from 50 to 90 ◦ C, the pH
from 4.0 to 8.0, while the reaction time was from 0.25 to 48 h. The
factorial design consisted of eight factorial points, six axial points
(two axial points on the axis of design variable), and four replications at the central point, leading to 18 experiments as shown in
Table 1. In each case, the CDs concentrations were determined by
HPLC.
The experimental design and analyses of results were carried out
using Statistica 13.0 (Statsoft, USA). Statistical analysis of the model
was performed as analysis of variance (ANOVA). The variance

explained by the model was given by the multiple determination
coefficients, R2 . For each variable, the quadratic models were represented as contour plots (2D).
2.8. Continuous production of CD in packed-bed reactor
The production of CDs was tested using a continuous packedbed reactor (Ø = 1 cm; height = 12 cm) with 1 g of silica with
immobilized enzyme. The reaction temperature was maintained
at 70 ◦ C by circulating water inside the jacket around the reactor. Substrate was 4% of soluble starch (w/v) and the pH was 4.0
(sodium acetate buffer, 10 mmol L−1 ) or pH 8.0 (sodium phosphate buffer, 10 mmol L−1 ). The substrate was fed through the
column using a peristaltic pump with variable flow rate, from
0.01 to 0.90 mL min−1 , representing residence times from 1.96 to
141.12 min. Samples were collected after reaching the steady state,
filtered at 0.22 ␮m Millipore membranes and analyzed by HPLC for
␣-, ˇ- and -CD quantification.
3. Results and discussion
3.1. Synthesis and characterization of support
Silica (hereafter assigned as Si) with controlled pore size was
synthesized using tetraethylorthosilicate (TEOS), which provides
highly reactive silanol groups on its surface. The material was functionalized by silanization with different concentrations of APTMS
(3-aminopropyltrimethoxysilane), incorporating amino groups to
the support (Si-NH). These amino groups were activated with
glutaraldehyde (Si-NH-G), enabling the covalent bond with the
enzyme.
Thermogravimetric analyses (TGA) were performed in order to
check the amount of organics incorporated in each step. The weigh
losses can be seen in the thermograms presented in Fig. 1. The
weight losses between 150 and 650 ◦ C represent the decomposition of APTMS (Pavan, Gobbi, Costa, & Benvenutti, 2002), and were
used to estimate the organic quantities. According to Table 2, the
silica was coated with increasing amounts of amino groups (0.29,
0.45 and 0.65 mmol g−1 of support), providing different activation
degrees for CGTase immobilization. The resulting modified silica

J.d.N. Schöffer et al. / Carbohydrate Polymers 169 (2017) 41–49

45

Table 4
Statistical analysis of the CCD for CDs production.
Variable

˛-CD

ˇ-CD

-CD

Effect

Standard error

p-Value

Effect

Standard error

p-Value

Effect

Standard error

p-Value

Mean

1.30a

0.03

<0.0001

0.86a

0.03

<0.0001

2.04a

0.05

<0.0001

Linear
X1
X2
X3

0.76a
0.65a

−1.40a

0.04
0.04
0.04

0.0002
0.0004
<0.0001

0.23a
1.09a
−1.51a

0.03
0.03
0.03

0.0065
<0.0001
<0.0001

0.23a
−0.56a
1.36a

0.05
0.05
0.05

0.0231
0.0019
0.0001

Quadratic
X1
X2
X3

1.32a
0.39a
0.04

0.04
0.04
0.04

<0.0001
0.0019
0.3608

0.98a
0.61a
−0.05

0.03
0.03
0.03

<0.0001

0.0004
0.2555

−0.49a
0.40a
−0.45a

0.05
0.05
0.05

0.0030
0.0057
0.0039

Interactions
X1 X2
X1 X3
X2 X3

−0.17a
−0.12
−0.22a

0.05
0.05
0.05

0.0388
0.0823

0.0192

−0.26a
0.07
−0.26a

0.04
0.04
0.04

0.0094
0.1870
0.0099

0.11
0.15
−0.15

0.07
0.07
0.07

0.2089
0.1145
0.1069

Variables X1 represent the temperature, X2 the pH and X3 the reaction time.
a
Statistically significant at 95% of confidence level.

supports were named as Si-NH-0.29, Si-NH-0.45 and Si-NH-0.65.
Characterization analyzes of the supports and derivatives were performed with lyophilized material.
The silica and its derivatives were texturally characterized
concerning the pore size distribution and N2 isotherms curves.
According to the results (N2 isotherms and BJH analysis presented
in the support material), it is possible to observe that the starting
silica is mesoporous having 24 nm as maximum of pore diameter
and surface area of 133 m2 g−1 . The functionalization with amino
groups leads to a slight decrease in the pore size, as well as, in the
surface area and pore volume (Table 2), with the increase of amino
group content. This behavior is in agreement with the increase of
the pore surface functionalization shown by TGA.
3.2. CGTase immobilization on mesoporous silica functionalized
with aminopropyl
The mesoporous silica functionalized using APTMS and activated with glutaraldehyde (Si-NH-G) was used to CGTase
immobilization. Using a protein load of 10 mg g−1 of support, the
immobilization yields were above 96%. However, the efficiency values were low, between 3 and 5% (Table 3). Silica with the highest
concentration of organic groups was able to bind larger amount
of enzyme, achieving higher yields and efficiencies of immobilization and thus higher enzyme activity per gram of support, reaching
to 10173 U g−1 . Low enzyme activity recovery is common in covalent immobilization of CGTases (Amud et al., 2008; Graebin et al.,
2016; Tardioli et al., 2006), perhaps by restricted access of the
high molecular weight substrate to the active site (Hernandez &
Fernandez-Lafuente, 2011). The advantage of using this biocatalyst in immobilized form stems from the possibility of reusing it in
batch or in continuous processes with better stability and operational control (Barbosa et al., 2015; Bolivar, Eisl, & Nidetzky, 2016;
Rodrigues et al., 2013; Sheldon, 2007). Because of the higher enzymatic activity per gram of support, the Si-NH-0.65-G was used for
the next experiments.
3.3. ˛-, ˇ- and -CDs production as a function of time
Initially, the production of ␣-, ˇ- and ␥-CD was tested using
free and immobilized CGTase (Si-NH-0.65-G), varying pH (4 and 7),
temperature (50 and 70 ◦ C) and reaction time (up to 48 h).

According to the results showed in Fig. 2, the production of
cyclodextrins was influenced by immobilization, temperature, pH,
and, especially by the reaction time. Since equivalent amounts of

free enzyme and immobilized enzyme were used, it is possible that
the profile and rate of cyclodextrin production are modulated by
the enzyme immobilization. This change may be due to the conformational change of the enzyme or even by longer contact time
between enzyme, substrate and products. At 1 h, the concentrations
of ␣- and ˇ-CDs were higher than -CD for free (2.5 mg mL−1 of ␣CD, 2.2 mg mL−1 of ˇ-CD and 1.3 mg mL−1 of -CD at 50 ◦ C and pH
7.0) and immobilized enzyme (3.9 mg mL−1 of ␣-CD, 3.4 mg mL−1 of
ˇ-CD and 0.7 mg mL−1 of -CD at 70 ◦ C and pH 7.0). However, along
the reaction time, the concentrations of ␣- and ˇ-CDs decreased,
increasing the -CD amount independent of the pH tested, reaching to 2.1 mg mL−1 for free (70 ◦ C, pH 7.0) and 2.96 mg mL−1 for
immobilized enzyme (70 ◦ C, pH 4.0).
Goel and Nene (1995) stated that the proportion of cyclodextrins produced by a CGTase from Bacillus firmus was dependent on
the initial substrate concentration and reaction time. The effects
of ionic species of buffers, pH values and reaction temperature on
®
the enzyme activity and product specificity of Toruzyme CGTase
were already mentioned by Kamaruddin et al. (2005). According to
the authors, when acetate buffer was used, comparable amounts
of ␣- and ˇ-CD were produced at the beginning, and after 1 h
of reaction, ␣-CD was slowly degraded and ˇ-CD was the main
cyclodextrin in the solution. Only a small amount of -CD was
detected at those conditions. However, when phosphate buffer was
used, after 6 h of incubation, the concentration of ␣- and ˇ-CD
decreased, but still with a small amount of -CD (Kamaruddin et al.,
2005). Additionally, Dura and Rosell (2016) also showed that the
pattern of products formed by CGTase is pH dependent. Initially, at
pH 6.0, only oligosaccharides were produced by the enzyme, and

CDs require longer incubation times to be formed. Whereas, at pH
4.0, CDs were formed in greater amounts, particularly ˇ-CD, at the
reaction begin.
Concerning the time, Tardioli et al. (2006) observed a decrease
in ˇ-CD concentration after 1 h of reaction using the immobilized
CGTase, probably because the CD was used for the production
of linear maltooligosaccharides. Decrease in the concentration of
cyclodextrins after a certain reaction time was also observed by
other authors, including CGTases from other sources, who indicated
that this is due to other reactions catalyzed by the enzyme in addition to the cyclization (Svensson, Ulvenlund, & Adlercreutz, 2009;
van der Veen, van Alebeek, Uitdehaag, Dijkstra, & Dijkhuizen, 2000).
It was also suggested that the use of the enzyme in continuous processes could avoid the degradation of the cyclodextrins due to the
continuous removal of the product (Tardioli et al., 2006; Wind et al.,
1995; Yang & Su, 1989; Zheng, Endo, & Zimmermann, 2002).


46

J.d.N. Schöffer et al. / Carbohydrate Polymers 169 (2017) 41–49

Fig. 3. Contour plots of CCD results. Columns: ␣-, ␤- and ␥-CD at different pH and time and at 50 ◦ C, 70 ◦ C and 90 ◦ C (lines).

3.4. Evaluation of ˛-, ˇ- and -CDs production using a central
composite design (CCD)
The previous experiments showed that the reaction conditions
affected the production of ␣-, ˇ- and -CDs. Thus, it was performed
a 23 central composite design (CCD) in order to evaluate the combined effect of temperature, pH and reaction time. The studied
conditions and the results for the experimental design are shown
in Table 1.
Linear, quadratic and interaction effects of the variables temperature, pH and reaction time are presented in Table 4. All linear

effects were statistically significant. Reaction time presented the
highest linear effect for all CDs, the negative effect of this variable for ␣- and ˇ-CD indicates that increasing the reaction time
there is a reduction in the concentration of both CDs. In contrast,
reaction time presented a positive effect for -CD. The pH showed
positive effect on ␣- and ˇ-CD production and a negative effect for
-CD, meaning that more alkaline pH favors ␣- and ˇ-CD production, while a larger amount of -CD will be produced under more
acidic conditions.
A comparative analysis of the amino acid sequence of different CGTases revealed a clear trend in product specificity based on
amino acids positioned in the vicinity of the active site. These amino
acids orient the substrate, playing crucial roles in the cleavage position of the glycosidic bond. Mutations in these positions modify
the interactions between neighboring amino acids, increasing or
decreasing the available space for the substrate and altering the

specificity of the enzyme (Kelly, Dijkhuizen, & Leemhuis, 2009).
Changes in the structure of the enzyme may also occur due to pH
and temperature variation and during the immobilization process
(Rodrigues et al., 2013), which may have been responsible for the
variation in CGTase specificity by varying the production of ␣-,
ˇ-, -CD and even linear products. Moreover, the immobilization
can be responsible by changes in the activity fixing the enzyme in
a specific conformation (Rodrigues et al., 2013). The relationship
between variables and responses can be better analyzed by examining the contour plots in Fig. 3. As can be seen, ␣- and ˇ-CDs are
simultaneously produced in higher concentrations at low reaction
times and mainly at alkaline pH and high temperatures. Whereas
the production of -CD showed a completely different behavior,
being produced at long reaction time, temperatures around 70 ◦ C
and lower pH, compared to ␣- and ˇ-CDs.
As the concentration of -CD increased (and ␣- and ˇCD decrease), the starch conversion rate in total cyclodextrins
decreased (data not shown). It could possibly be by the use of ␣and ˇ-CDs as substrates, because CGTase is capable of catalyzing the
hydrolysis of ␣(1-4) bonds and disproportionation, besides of the

cyclization and transglycosylation reactions. It has been reported
the ability of CGTase to firstly produce cyclodextrins of larger sizes
and then to reduce them (Terada, Yanase, Takata, Takaha, & Okada,
1997), but there are no reports of the initial production of smaller
CDs and then increase them.
de Souza et al. (2013) proposed a kinetic model for cyclodextrins
production considering the reversibility of the cyclization and other


J.d.N. Schöffer et al. / Carbohydrate Polymers 169 (2017) 41–49

47

Fig. 4. Continuous production of (᭹) ␣-CD, (᭿) ␤-CD, ( ) ␥-CD at 70 ◦ C, pH 8.0 (a) and pH 4.0 (b), on a packed-bed reactor with immobilized CGTase.

side reactions at the same time. According to the authors, the disproportionation reaction is generally much faster than the others,
leading rapidly to a state of equilibrium with the linear dextrins,
which will be cyclized by the enzyme. In addition, there are several
studies using ␣-CD as substrate for the formation of other products
than CDs (Mathew & Adlercreutz, 2012; Svensson & Adlercreutz,
2011; Zehentgruber, Lundemo, Svensson, & Adlercreutz, 2011).
Thus, ␣- and ˇ-CD could be hydrolyzed and used in disproportionation reaction, forming larger linear dextrins, which could be
further cyclized to form -CD. The preference in using ˛- and ˇCD, rather than -CD, as a substrate may be because -CDs assume
more flexible and not regular cylinder shaped forms, almost as a
collapsed structure described by Szejtli (1998) for ␦-CD (9 glucose
units), making it difficult to recognize and use by the enzyme.
Therefore, it is clear the possibility of modulating the production
of these three cyclodextrins, using the same immobilized CGTase,
only by varying the reaction conditions. The possibility of directing
the production to obtain higher concentrations of -CD is of particular interest, since its industrial production is based on the use

of complexing agents, which form insoluble inclusion compounds
and extract it selectively. Furthermore, the process of its separation
and purification are expensive and time consuming (Li et al., 2007;
Wang, Wu, Chen, & Wu, 2013).
Among the treatments, ␣- and ˇ-CDs presented the lowest
productions at 70 ◦ C, pH 6.0 and 48 h (0.72 and 0.14 mg mL−1 ,
respectively) (values presented in Table 1). At 90 ◦ C, pH 6.0 and
24 h the highest concentration of ␣-CD production was obtained
(4.93 mg mL−1 ), while for ˇ-CD, it was obtained at 58 ◦ C, pH 7.2
and 9.9 h (3.63 mg mL−1 ). On the other hand, the highest -CD production was at pH 4.0, 70 ◦ C and 24 h (3.52 mg mL−1 ).
3.5. Modulated production of ˛-, ˇ- and -CD on continuous
reactor
Heterogeneous biocatalysts provide greater stability and
enables them to be used in continuous systems, and in this way, the
benefits of this mode of processing can be explored: the increase of
productivity and the consequent reduction of the costs of preparation and use of the same, enabling processes. Therefore, the
production of ␣-, ˇ- and -CD was tested at continuous reaction in
a packed-bed reactor. As the reaction temperature was the parameter that had the lowest effect on the CDs production, this was
kept constant and once the enzyme is thermostable, the operating temperature of the reactor was maintained at 70 ◦ C to facilitate

the substrate solubility. Based on the previous experiments, two
conditions were tested, aiming to direct the CD production. For ␣and ˇ-CD, it was used pH 8.0 and higher flow rates (low residence
time), and for the directed production of ␥-CD, it was used pH 4.0
and smaller flow rates (high residence time). The reactor was filled
with 1 g immobilized CGTase and soluble starch (4% w/v) was used
as substrate.
The results presented in Fig. 4, show the importance of reaction
time for directed production of ␣-, ˇ- and -CD.
At pH 8.0 (Fig. 4a), the lower residence times favor the production of ␣- and ˇ-CD, reaching to 4.3 (␣-CD) and 4.9 (ˇ-CD) mg mL−1
at 6.72 min of reaction. At pH 4.0 and 7.47 min the highest starch

conversion in cyclodextrins (26%) was obtained. At this pH condition, higher residence (141 min) time leads to a decreased of ␣- and
ˇ-CD concentrations from 4.6 (for both) to 0.75 mg mL−1 (␣-CD)
and 0 mg mL−1 (ˇ-CD) (Fig. 4b), whereas the -CD concentration
increased from 1.2 to 3.4 mg mL−1 . These results confirm what was
observed in the experimental design, where the reaction time presented the highest effect, being negative for ␣- and ˇ-, and positive
for -CD production.
Using a CGTase immobilized in a mixed gel beads by entrapment, Rakmai and Cheirsilp (2016) tested the ˇ-CD production in
a comparative study in continuous stirred tank reactor (CSTR) and
packed-bed reactor (PBR). Using soluble starch at 4%, pH 7.0 and
50 ◦ C they reached to 4.64 mg mL−1 of ˇ-CD on PBR reactor and
6.10 mg mL−1 on CSTR. The authors observed that even increasing the amount of enzyme in the reactor, there was a limit from
which the ˇ-CD production remained constant. However, there
was an increase in reducing sugar concentration, probably due to
hydrolytic activity of the enzyme.
Our results showed that it is possible to modulate the CDs production using two different conditions, increasing ␣- and ˇ-CD
by one side, and -CD by the other, thus facilitating the purification steps. ␣- and ˇ-CD are preferentially produced at pH 8.0 and
1.96 min, obtaining, respectively, 3.44 mg mL−1 and 3.51 mg mL−1
with only 0.62 mg mL−1 of -CD, while at pH 4.0 and 141 min, -CD
was produced as the main product, with concentrations of 0.75 and
3.36 mg mL−1 , for ␣- and -CD respectively.
4. Conclusion
In this study, we immobilized a CGTase on mesoporous silica functionalized with different amounts of aminopropyl groups
and activated with glutaraldehyde, obtaining higher efficiency with


48

J.d.N. Schöffer et al. / Carbohydrate Polymers 169 (2017) 41–49

greater amounts of organic groups on the support. The immobilized biocatalyst was used in an experimental design, evaluating

the effect of pH, temperature and reaction time in the production of cyclodextrins. The results showed the importance of these
parameters for directed production of ␣-, ˇ- and -CD. ␣- and ˇ-CD
were preferably produced at more alkaline pH and in short reaction time. While the production of -CD is favored in more acidic
pHs and longer times. The possibility of modulating the production of cyclodextrins was also identified on continuous production
system in a fixed bed reactor. In this context, this work presented
the possibility of using the same immobilized CGTase to produce
the cyclodextrin of interest, especially for -CD, which has great
industrial importance. Moreover, the immobilized enzyme allows
a continuous use, which greatly facilitates the process control, productivity and purification steps.
Acknowledgements
This work was supported by the Coordenac¸ão de
Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), Fundac¸ão
de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS)
and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) of the Brazilian government.
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