An innovative method for immobilizing sucrose isomerase on ε-poly-L-lysine modified mesoporous TiO2

Article abstract of DOI:10.1016/j.foodchem.2015.04.072

Sucrose isomerase (SIase) is the key enzyme in the enzymatic synthesis of isomaltulose. Mesoporous titanium dioxide (M-TiO₂) and ε-poly-L-lysine-functionalized M-TiO₂ (EPL-M-TiO₂) were prepared as carriers for immobilizing SIase. SIase was effectively immobilized on EPL-M-TiO₂ (SI-EPL-M-TiO₂) with an enzyme activity of 39.41 U/g, and the enzymatic activity recovery rate up to 93.26%. The optimal pH and temperature of immobilized SIase were 6.0 and 30 °C, respectively. SI-EPL-M-TiO₂ was more stable in pH and thermal tests than SIase immobilized on M-TiO₂ and free SIase. K_m of SI-EPL-M-TiO₂ was 204.92 mmol/L, and ν_max was 45.7 μmol/L/s. Batch catalysis reaction of sucrose by SI-EPL-M-TiO₂ was performed under the optimal conditions. The half-life period of SI-EPL-M-TiO₂ under continuous reaction was 114 h, and the conversion rate of sucrose after 16 batches consistently remained at around 95%, which indicates that SI-EPL-M-TiO₂ has good operational stability. Thus, SI-EPL-M-TiO₂ can be used as a biocatalyst in food industries.

Full text of DOI:10.1016/j.foodchem.2015.04.072

Food Chemistry 187 (2015) 182–188
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
An innovative method for immobilizing sucrose isomerase
on -poly-L-lysine modified mesoporous TiO2
e
⇑
⇑
Lingtian Wu, Yi Liu, Bo Chi, Zheng Xu, Xiaohai Feng, Sha Li , Hong Xu
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Food Science and Light Industry, Nanjing Tech University, 30 Puzhu South Road, Nanjing 211816, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Sucrose isomerase (SIase) is the key enzyme in the enzymatic synthesis of isomaltulose. Mesoporous tita-
Received 8 October 2014
Received in revised form 11 April 2015
Accepted 17 April 2015
nium dioxide (M-TiO
for immobilizing SIase. SIase was effectively immobilized on EPL-M-TiO
enzyme activity of 39.41 U/g, and the enzymatic activity recovery rate up to 93.26%. The optimal pH
and temperature of immobilized SIase were 6.0 and 30 °C, respectively. SI-EPL-M-TiO was more stable
in pH and thermal tests than SIase immobilized on M-TiO and free SIase. K of SI-EPL-M-TiO
04.92 mmol/L, and vmax was 45.7 mol/L/s. Batch catalysis reaction of sucrose by SI-EPL-M-TiO
performed under the optimal conditions. The half-life period of SI-EPL-M-TiO
was 114 h, and the conversion rate of sucrose after 16 batches consistently remained at around 95%,
2
) and
e
-poly-
L
-lysine-functionalized M-TiO
2
(EPL-M-TiO
2
) were prepared as carriers
2
(SI-EPL-M-TiO ) with an
2
Available online 23 April 2015
2
2
m
2
was
was
Chemical compounds studied in this article:
Titanium dioxide (PubChem CID: 26042)
Sucrose (PubChem CID: 5988)
Isomaltulose (PubChem CID: 439559)
Trehalulose (PubChem CID: 162104)
2
l
2
2
under continuous reaction
which indicates that SI-EPL-M-TiO
a biocatalyst in food industries.
2
has good operational stability. Thus, SI-EPL-M-TiO
2
can be used as
Keywords:
Ó 2015 Published by Elsevier Ltd.
M-TiO
Sucrose isomerase
-Poly- -lysine
2
e
L
Isomaltulose
Immobilization
1
. Introduction
parenteral nutrient acceptable to diabetics (Hamada, 2002;
Kawai, Okuda, & Yamashita, 1985).
Isomaltulose
(
a
-
D
-glucosylpyranosyl-1,6-
D
-fructofuranose),
Recently, the enzymatic conversion of sucrose to isomaltulose
has drawn wide attention using sucrose isomerase (SIase, EC
5.4.99.11), found in many strains. The SIase with molecular weight
commonly referred to as palatinose, is a potential disaccharide
sugar substitute and structural isomer of sucrose. Isomaltulose is
found in small quantities in honey and sugar cane. Isomaltulose
and its hydrogenated derivative, isomaltitol, have attracted world-
wide attention because of their function as a food ingredient (Li
et al., 2013; Ravaud, Watzlawick, Haser, Mattes, & Aghajari,
of 66 kDa hydrolyzes the
and then forms an -1,6 bond to produce isomaltulose or an
bond to produce trehalulose (1-O- -glucopyranosyl- -fructose)
a-1,2 bond between glucose and fructose
a
a-1,1
a-D
D
(Cheetham, 1984; Huang, Hsu, & Su, 1998; Véronèse & Perlot,
1999). Several bacterial strains have recently been investigated
for isomaltulose production by cell immobilization. The immobi-
lized recombinant Escherichia coli cells producing SIase exhibited
good stability for 40 days with 83% ± 2% isomaltulose yield (Li
et al., 2013). Krastanov, Blazheva, and Stanchev (2007) and
Krastanov and Yoshida (2003) reported a 90% sucrose conversion
with immobilized Serratia plymuthica cells. De Oliva-Neto and
Menao (2009) reported that the specific activity of the immobilized
Protaminobacter rubrum cells ranged from 1.6 g to 4.0 g isomal-
tulose g/pellet/h with 70% and 65% (w/v) sucrose solution, respec-
tively. Li, Zhao, An, and Zhang (2003) observed that the
immobilized Klebsiella sp. LX3 cells could convert 99.7% of 10%
(w/v) sucrose solution into 87% isomaltulose, but the conversion
2
006). Isomaltulose is slowly hydrolyzed by isomaltase and
absorbed as glucose and fructose in organism (Haberer, Thibault,
Langhans, & Geary, 2009). Contrary to sucrose, isomaltulose can
also prevent tooth decay, attenuate insulin levels and the glycemic
index in the bloodstream, which indicates its potential as a
Abbreviations: SIase, sucrose isomerase; EPL,
porous titanium dioxide; EPL-M-TiO -poly- -lysine-functionalized mesoporous
titanium dioxide; SI-M-TiO sucrose isomerase immobilized on mesoporous
titanium dioxide; SI-EPL-M-TiO sucrose isomerase immobilized on -poly-
lysine-functionalized mesoporous titanium dioxide.
Corresponding authors. Tel./fax: +86 25 58139433.
E-mail addresses: lisha@njtech.edu.cn (S. Li), xuh@njtech.edu.cn (H. Xu).
2
e-poly-L-lysine; M-TiO , meso-
2
,
e
L
2
,
2
,
e
L-
⇑
http://dx.doi.org/10.1016/j.foodchem.2015.04.072
308-8146/Ó 2015 Published by Elsevier Ltd.
0

L. Wu et al. / Food Chemistry 187 (2015) 182–188
183
rate of sucrose decreased to 71.8% when the sucrose concentration
2.2. Production and purification of SIase
was increased up to 30%. The above immobilized cells, however,
also exhibit some deficiencies. For example, space steric hindrance
increased compared with free enzyme or cells, and the unit
enzyme activity could catalyze fewer substrates. To a certain
extent, enzyme immobilization techniques can solve the above
problems, but few studies have reported on immobilization of
the SIase for the conversion of sucrose to isomaltulose.
Enzyme immobilization techniques can be employed to sepa-
rate enzymes from the reaction medium easily by simple sedimen-
tation and facilitate the continuous reaction (Fernandez-Lafuente,
SIase from E. coli (pET22b-palI) using fermentation medium,
induced with 0.5 mM lactose (Li et al., 2011). The cells were
decomposed by ultrasonic, carried out in buffer A (10 mM Hepes,
500 mM NaCl, and 10% glycerol, pH 8.0), after washed with buffer
B (50 mM, pH 6.0 phosphate buffer). Then cell disruption liquid
was centrifuged to remove the debris of cells. A Ni–NTA resin col-
umn was used to load the filtrate, which was equilibrated with
buffer A. Then the column was washed by the buffer A containing
50 mM imidazole, and a gradient of imidazole (from 100 mM to
300 mM) was applied to elute enzymes. The enzyme solution
was dialyzed overnight against buffer B with 10% glycerol, and
the dialysate was stored at 4 °C. The purity of SIase was deter-
mined by SDS–PAGE analysis.
2
009). More importantly, it also has the potential to improve sta-
bilities and reuse enzymes in numerous bioreactor applications
Cisani, Varaldo, Ingianni, Pompei, & Satta, 1984; Kim, Grate, &
(
Wang, 2006; Moreno & Sinisterra, 1994).
In recent years, many mesoporous materials are popularly used
as carriers for enzyme immobilization. Of them, mesoporous tita-
2 2
2.3. Preparation of enzyme carriers M-TiO and ELP-M-TiO
2
nium dioxide (M-TiO ), one type of mesoporous materials, is an
environmentally friendly and highly stable nanocrystalline mate-
rial. This material has high surface area-to-volume ratio, good bio-
compatibility and coordination ability with amine and carboxyl
groups (Lori & Nok, 2004; Sadjadi, Farhadyar, & Zare, 2009; Tang,
Yan, Tai, & Chan, 2010; Wei, Lu, Ouyang, Yong, & Yu, 2013).
The M-TiO
dispersed for 30 min. Then, the M-TiO
using vacuum suction filtration. The M-TiO
persed in 40 mL buffer B with 3.0% (w/w) EPL. The mixture was
incubated at 25 °C for 10 h. After filtration, the powders (EPL-M-
2
powder was mixed with buffer B and ultrasonically
powder was separated
powder (1 g) was dis-
2
2
These unique properties of M-TiO
enzyme immobilization or attachment of enzymes in enzymatic
bioreactors. The enzymes can be combined onto the inner and an
2
make it an ideal carrier for
TiO
2.4. Immobilization of SIase on M-TiO
1 g M-TiO (or EPL-M-TiO ) powders were put in different con-
centrations of 40 mL SIase solution (0.15–1.35 U/mL), respectively.
The mixture was then stirred at 25 °C for 6 h. Then, the enzyme to
2
) were washed thrice with buffer B.
2
and EPL-M-TiO
2
2
outer surface of M-TiO , and the amount of enzymes attached to
outer surface of carrier relative to that attached to the inside sur-
face of the carrier is negligible (Kotha, Raman, Ponrathnam,
Kumar, & Shewale, 1998).
2
2
Enzymes immobilized on supports have been considered to
occur through electrostatic binding, simple adsorption of enzymes
onto their surface, and covalent attachment or encapsulation,
which has achieved relatively satisfactory results. Wei et al.
be immobilized on M-TiO
2 2 2
or EPL-M-TiO (SI-M-TiO or SI-EPL-M-
TiO ) was separated using vacuum suction filtration, and the filter
2
liquor was preserved for the determination of enzyme activity. To
remove the untrapped enzyme molecules, the immobilized
enzymes were washed with buffer B until no SIase activity could
be detected in washings. The washings were collected to estimate
the amount of enzyme bound on the carriers. The percentage of
immobilized SIase activity to initial SIase activity in solution is
defined as the enzymatic activity recovery.
(
2013) reported the immobilization of b-glucosidase on mercapto-
propyl functionalized M-TiO . After 10 batches hydrolysis, the cel-
lobiose conversion rates were still around 90%. Wang et al. (2014)
chemically modified M-TiO with 3-aminopropyltriethoxysilane,
and the immobilization -glutamyltranspeptidase exhibited good
2
2
c
operational stability. In addition to the above enzymes, as well as
alkaline protease and bone morphogenetic protein were immobi-
2 2
2.5. Structural characteristics of M-TiO and EPL-M-TiO
lized on TiO
et al., 2009).
2
successfully (Han, Jang, Kim, & Koh, 2014; Sadjadi
Scanning electron microscopy (SEM, S-4800, Hitachi, Japan) was
used to investigate the surface morphology of M-TiO and EPL-M-
TiO . Fourier-transform infrared spectroscopy (FTIR, PerkinElmer
Spectrum 100 instrument) was conducted to determine the combi-
In this paper we investigated the immobilization of SIase on M-
TiO and -poly- -lysine-mesoporous titanium oxide (EPL-M-TiO ),
2
2
e
L
2
2
the biochemical characteristics of free and immobilized enzyme
and the conversion of sucrose to isomaltulose and trehalulose.
Therefore, a rational strategy of a more specific and efficient immo-
bilized enzyme for economical production of isomaltulose in food
industry is provided. Previous studies (Huang et al., 1998; Lee,
Kim, Kim, & Lee, 2008; Li et al., 2013) had shown that isomaltulose
was always produced with SIase in the form of free enzyme or
immobilized cells. This study successfully immobilized the SIase
nation of EPL and M-TiO
2
.
2.6. Enzyme assay
2.6.1. Free enzyme
The reaction mixture was composed of 0.8 mL 50 g/L (w/v)
sucrose in buffer B and 0.2 mL of appropriately diluted SIase. The
reaction was incubated at 30 °C for 20 min, which was stopped
by boiling for 10 min, and centrifuged at 12,000Âg for 5 min to
remove denatured proteins. The amount of isomaltulose in the
supernatant was determined by high-performance liquid chro-
matography (HPLC, Agilent 1200, USA system equipped with a
refractive index detector). One unit (U) of SIase activity is defined
on the surface of EPL-M-TiO
bution to the immobilization technology.
2
, and it is expected to make its contri-
2
. Materials and methods
2.1. Materials
as the amount of enzyme that forms 1 lmol of isomaltulose per
minute under standard assay conditions.
EPL was produced by Streptomyces albulus PD-1 strain, with a
molecular weight of approximately 2.5–3.5 kDa (Xia, Xu, Feng,
Xu, & Chi, 2013). M-TiO was provided by Lu (He, Lu, Feng, Yu, &
Yang, 2004). The other reagents and chemicals used in this study
were purchased from Aladdin Industry Co., Ltd. (Shanghai, China).
2.6.2. Immobilized enzyme
2
Immobilized enzyme activity was measured by incubating
0.025 g of immobilized enzyme with 1 mL of 40 g/L (w/v) sucrose
in buffer B at 30 °C for 20 min and stopped by centrifuging at

1
84
L. Wu et al. / Food Chemistry 187 (2015) 182–188
1
2,000Âg for 1 min. Thus, 1 U of immobilized enzyme activity is
2
3.2. M-TiO modified with EPL
defined as the amount of SIase that releases 1
per minute under standard assay conditions.
lmol isomaltulose
Surface of M-TiO
2
is lined with dense hydroxyl groups, which
to carry negative charges, under
provides the support of M-TiO
2
physiological pH conditions (Wang et al., 2014). Due to the pres-
ence of abundant free amino groups, EPL carries positive charges
2.7. Sugar composition
(
(
Xia et al., 2013), whereas SIase also carries negative charges
Ravaud et al., 2007). Therefore, the modified M-TiO was embed-
Prior to HPLC analysis, the reaction mixture was filtered by
.22 membrane filters. The samples were appropriately
2
0
lm
ded by EPL, changed the characteristics of the surface of the M-TiO
with negative charges. The above indicated that EPL, one of the
polycation biological materials, was used to modify M-TiO in
physiological pH solutions, which could not only improve the bio-
compatibility between enzyme and M-TiO , but also increase the
amount of enzyme loading on the surface of M-TiO due to the
electrostatic adsorption between positive and negative charges.
Thus, it could reduce the resistance of combination between
enzyme and substrate. All the above characteristics were attribu-
ted to microenvironmental effect, under the existence of EPL, the
microenvironment of enzyme changed (Venkataraman, Horbett,
& Hoffman, 1977).
2
diluted, and 20
Pb column (10
l
l
L diluted sample was injected onto a BP-100
m, 300 mm  7.8 mm) to measure sugar compo-
2
+
2
sition. The mobile phase was HPLC grade water with a flow rate of
0
.4 mL/min at 80 °C. Sugar components were analyzed with a
2
+
2
refractive index detector (RI-101). The HPLC and BP-100 Pb col-
umn were purchased from Agilent (Wilmington, DE) and Benson
2
(
Beijing, China).
2
.8. Properties of immobilized and free enzymes
The optimal temperature of free and immobilized enzymes was
investigated from 10 °C to 60 °C at pH 6.0. The thermal stability of
SIase was evaluated by incubating the enzyme at different temper-
atures (10–60 °C) at pH 6.0 for 4 h. To determine the optimal pH,
enzyme assay was investigated from pH 4.5 to pH 8.0 in citric
acid-phosphate buffer at 30 °C. Similarly, the pH stability of SIase
was examined by incubating the enzyme at different pH levels
3.3. Structural characteristics of M-TiO and EPL-M-TiO
2
2
M-TiO2 was characterized by SEM and FTIR before and after
being functionalized, respectively. In Fig. 1, SEM results of the sur-
face morphologies analysis of M-TiO and EPL-M-TiO were shown.
2
2
(
4.5–8.0) at 4 °C for 36 h. Unless specified, all reactions were per-
By contrast, after modification, the shape of the surface has chan-
ged significantly, and EPLs in the form of social covering were
observed on the surface of M-TiO2.
formed as the method of enzyme assay. The residual activity of free
and immobilized enzyme was estimated under standard assay
conditions.
FTIR spectra were recorded for native and EPL treated M-TiO to
2
2
understand the interaction between EPL and M-TiO (Fig. 2). The
FTIR spectra shows the characteristic absorption peak of Ti–O–Ti
2
.9. Determination of kinetic parameters
À1
at approximately 495 cm , and the stretching vibration peak near
À1
3
2
1
419 cm is due to the hydroxyl group of M-TiO
2
(Wang et al.,
The kinetic parameters of immobilized and free enzyme were
014). The C@O stretch vibration of amide bond is near
determined by measuring the rate of reaction with an increasing
concentration of sucrose (35–840 mM). The reactions were con-
ducted in buffer B, at 30 °C for 20 min. The Michaelis–Menten
À1
À1
668 cm . The peak at 1537 cm corresponds to CANAH stretch-
À1
ing vibration of amido bond, whereas the peak at 1100 cm shows
the CAN stretching of the amido bond (Liang, Yuan, Liu, Wang, &
Constant (K
m
) and maximum reaction velocity (vmax) values were
Gao, 2014; Shima & Sakai, 1977). The peak at approximately
calculated using the Lineweaver–Burk equation.
À1
3
150 cm shows the NAH stretching that corresponds to the free
amino group (Rao et al., 2012). The SEM and FTIR results show the
existence of amine group and amido bond at the carrier surface
2.10. Reusability of the immobilized enzymes
and also indicate that EPL has been combined with M-TiO
successfully.
2
,
The batch reaction was performed with 20 mL sucrose solution
(
2 2
50 g/L) and 0.5 g SI-M-TiO (or SI-EPL-M-TiO ) in a 50 mL
Erlenmeyer flask. The reactions were incubated in a shaking bath
3.4. Immobilization of SIase on M-TiO and EPL-M-TiO
2
2
at 30 °C until the substrate was completely catalyzed to products.
SI-M-TiO
2
and SI-EPL-M-TiO
2
were used repeatedly, which were
2 2
SIase was immobilized on M-TiO and EPL-M-TiO at the initial
washed with buffer B after every batch. All samples of every batch
were analyzed by HPLC after incubation.
concentration of 0.15 U/mL to 1.35 U/mL free SIase in solution.
With the increase in the initial enzyme concentration, the activity
2
of SI-M-TiO gradually increased (Table 1). However, the enzyme
activity recovery rate declined precipitously. When the initial
enzyme concentration was greater than 0.75 U/mL, the activity of
2.11. Statistical analysis
SI-M-TiO
2
did not show obvious difference with the increase in
activity was basi-
Data were expressed as the means ± standard deviation.
Statistical analysis was conducted by one-way ANOVA followed
by Duncan’s test (P < 0.05) using the SPSS 17.0 software.
the initial enzyme concentration, and SI-M-TiO
cally stable at about 8.2 U/g. Inversely, increasing the initial
enzyme concentration resulted in a rapid increase in the activity
2
of SI-EPL-M-TiO
1.05 U/mL, SI-EPL-M-TiO
However, further increase in the initial concentration of enzyme
sharply reduced SI-EPL-M-TiO activity. This phenomenon is
2
.
When the initial SIase concentration was
3
. Results and discussion
2
activity reached up to 39.41 U/g.
3.1. Purification of the recombinant SIase
2
caused by the increase in the amount of enzyme loading on the
carrier, which causes the space steric effect. Caldwell, Axen,
Bergwall, Olsson, and Porath (1976) reported that the activity of
immobilized enzyme was determined by the load of enzymes on
the carriers without space steric hindrance. Additionally, the
SDS–PAGE analysis of purified proteins revealed a single
polypeptide with the presence of abundant protein around
6 kDa (Data not shown). After enrichment, the activity of purified
enzyme was 128.4 U/mL under optimal conditions.
6

L. Wu et al. / Food Chemistry 187 (2015) 182–188
185
2 2
Fig. 1. SEM images of (a) M-TiO and (b) EPL-M-TiO .
2
the EPL changed the nature of the charges at the surface of M-TiO ,
which could avoid the repulsion between the same charges and
prompt the electrostatic adsorption between different charges.
Furthermore, EPL can reduce the risk of microbial contamination
of sugar solution during isomaltulose production from sucrose
(Bo et al., 2014).
3
3
.5. The properties of immobilized and free SIase
.5.1. Optimal pH and temperature
The optimization of pH is a significant step for increasing the
activity of both immobilized and free enzymes. The pH-dependent
activities of immobilized and free enzyme were determined at pH
4
.5 to 8.0 (Fig. 3a). As is shown no significant changes are observed
at the optimal pH (6.0) of free and immobilized SIase. However,
SIase immobilized by EPL-M-TiO had a wider pH range. The effect
2
of temperature on enzyme activity of immobilized and free
enzymes were performed from 10 °C to 60 °C (Fig. 3c). The optimal
temperature for the reaction of three enzymes was 30 °C.
Compared with that of the other two enzymes, the activity of SI-
2 2
Fig. 2. FTIR spectra of (a) M-TiO and (b) EPL-M-TiO .
electrostatic interaction among the enzymes probably changed the
conformation of enzyme. EPL-M-TiO , the EPL functionalized car-
rier, showed higher enzymatic activity recovery than the native
M-TiO . The highest enzymatic activity recovery of SI-M-TiO
was 83.75% at initial SIase concentration of 0.15 U/mL. The highest
enzymatic activity recovery of SI-EPL-M-TiO was 93.26% at initial
EPL-M-TiO
broadening of the optimal range. From 15 to 45 °C, the relative
activity of SI-EPL-M-TiO was more than 90%. For isomaltulose pro-
2
was more active at high temperature with noticeable
2
2
2
2
duction from sucrose, isomerization reaction conducted from 30 °C
to 40 °C is the most suitable temperature for industrial isomal-
tulose production, because the physiological temperature do not
cause the inactivation of enzyme and can save energy. Compared
with other catalytic reaction temperature, 40 °C is a relatively high
temperature, which is beneficial to the production of isomaltulose
2
SIase concentration of 1.05 U/mL. Selecting the highest enzymatic
activity recovery of immobilized operation, the activity of SI-EPL-
M-TiO
M-TiO
2
(39.41 U/g) was approximately eightfold as much as SI-
(5.04 U/g). It was obvious that the amount of enzyme load-
2
(Li & Xu, 2011). When the temperature was 60 °C, the relative
ings on M-TiO
ducive to the immobilization of SIase for its good heat stability,
water solubility and unique structure linking -carboxylic and e-
2 2
and EPL-M-TiO was very remarkable. EPL is con-
activity still maintained more than 70%. However, the activity of
free enzyme was almost denatured at 60 °C. These results show
that the range of optimal pH and temperature has been improved,
a
amino groups, especially the free amino groups it contains (Xia
et al., 2013; Zhang et al., 2012). Thus, the EPL can simultaneously
improve the biocompatibility between carriers and enzymes and
hence enhance their adhesion strength. So the above indicated that
2
compared with free SIase and SI-M-TiO (Fig. 3c). Furthermore, pH
6
.0 as the optimal pH of immobilized SIase meets the requirement
of industry to maintain the pH value within 6.0–6.5, which can
remarkably control the browning changes (Rhimi et al., 2009).
Table 1
Effect of the initial enzyme concentration on immobilized SIase.
Initial SI activity in the free SI solution (U/mL)
Immobilized enzyme activity (U/g)
Activity in the supernatant (%)
Actual enzymatic activity
recovery rate (%)
SI-M-TiO
2
SI-EPL-M-TiO
2
SI-M-TiO
2
SI-EPL-M-TiO
2
SI-M-TiO
2
SI-EPL-M-TiO
2
0.15
0.45
0.75
1.05
1.35
5.04 ± 0.12a
7.21 ± 0.52b
8.21 ± 0.77bcd
8.46 ± 0.08c
8.70 ± 0.05d
3.75 ± 0.01a
11.71 ± 0.12b
20.48 ± 1.61c
39.41 ± 0.08e
35.69 ± 0.52d
14.14 ± 0.09a
59.02 ± 0.41b
71.09 ± 1.21c
77.42 ± 0.25d
80.79 ± 3.63d
35.68 ± 1.9c
33.44 ± 2.4bc
30.70 ± 0.8b
4.04 ± 0.01a
31.42 ± 0.11b
83.75 ± 0.51e
39.44 ± 0.18d
27.04 ± 0.51c
19.34 ± 0.10b
15.57 ± 0.06a
61.28 ± 0.21a
64.70 ± 0.35b
67.94 ± 1.91c
93.26 ± 0.33d
65.75 ± 1.00bc
Each value represents the mean ± standard deviation. Different lowercase letters of each column indicate significant difference at P < 0.05.

1
86
L. Wu et al. / Food Chemistry 187 (2015) 182–188
Fig. 3. Effects of (a) pH and (c) temperature on the activity of immobilized and free SIase. (b) pH stability of immobilized and free SIase. (d) Thermal stability of immobilized
and free SIase. (j) Free SIase, (d) SI-M-TiO and (N) SI-EPL-M-TiO . The highest activity was defined as 100%.
2
2
The wide pH and temperature ranges offer elasticity of operation at
the industrial production. And the optional pH and temperature
stable than free SIase, maintaining more than 95% activity at pH
4.8–7.2. However, free SIase retained 95% activity at pH 5.8–6.3,
which is a narrow pH range. These results show that the range of
make the SI-EPL-M-TiO
maltulose production.
2
a desirable immobilized enzyme for iso-
thermal and pH stability of SIase immobilized on EPL-M-TiO
expanded to a certain degree (Fig. 3b).
2
is
3
.5.2. Thermal and pH stability
Thermal stability was tested by measuring the activity after
incubation at pH 6.0 from 10 to 60 °C for 4 h. As shown in
Fig. 3d, the immobilized (SI-M-TiO and SI-EPL-M-TiO ) and free
enzyme show similar stability at 10–20 °C. However, at incubation
temperatures over 20 °C, the thermal stability of SI-EPL-M-TiO
was better than that of free SIase and SI-M-TiO . Moreover, the
residual activity of SI-EPL-M-TiO still remained 60% after incuba-
tion at 50 °C for 4 h. However, the others were both below 30%.
Increasing the reaction temperature can remarkably increase the
reaction rate. The carriers are often helpful in improving the stabil-
ity of the enzyme with a hydrophilic environment (Hwang et al.,
3.6. Kinetic parameters determination
2
2
Km and vmax values were determined using Lineweaver–Burk
equation by varying the sucrose concentrations at the same tem-
perature and pH. The SI-EPL-M-TiO2 shows the lowest Km value
in the three enzymes (Table 2). This indicates that SI-EPL-M-TiO2
has the highest affinity for sucrose, and the vmax value matches free
SIase. Compared with free SIase, the affinity for sucrose of SI-M-
TiO2 decreases sharply, and the vmax is also slightly reduced. The
decreased Km value for SI-EPL-M-TiO2 indicates that EPL may
induce SIase directional immobilization on carriers and EPL may
make the enzyme active site contact with the substrate rapidly,
as well as the product can quickly leave. Given that SIase entered
2
2
2
2
004). EPL contains extensive hydrophilic groups (amino groups),
which provides the hydrophilic environment for enzyme immobi-
lization. EPL-M-TiO provides a high amino density for an efficient
2
immobilization of SIase through many binding sites. SIase is most
likely associated with direct interactions between the EPL and the
SIase, which can stabilize its native conformation, increase the con-
formational rigidity of SIase and protect the SIase against denatu-
ration at a higher temperature (Mahmoud, Lam, Hrapovic, &
Luong, 2013). The stability of different pH values was determined
Table 2
Apparent kinetic parameters of immobilized and free SIase.
Types of enzyme
Free enzyme
SI-M-TiO
SI-EPL-M-TiO
K
m
(mmol/L)
max
v (lmol/L/s)
259.71 ± 7.78b
435.55 ± 20.39c
204.92 ± 4.30a
46.03 ± 0.87b
32.14 ± 1.93a
45.71 ± 1.46b
2
with free SIase, SI-M-TiO
residual activity of SIase after incubation at pH 4.5 to 8.0 for
6 h. As shown in Fig. 3b, SI-M-TiO and SI-EPL-M-TiO were more
2
2
and SI-EPL-M-TiO by measuring the
2
Each value represents the mean ± standard deviation. Different lowercase letters of
each column indicate significant difference at P < 0.05.
3
2
2

L. Wu et al. / Food Chemistry 187 (2015) 182–188
187
Fig. 4. Number of cycles of the immobilized enzymes that catalyzed sucrose into isomaltulose and trehalulose. (h) SI-EPL-M-TiO
2
and (j) SI-M-TiO
2
. The first sucrose
conversion rate was defined as 100%.
the channel of M-TiO
max value for SI-M-TiO
drance and diffusion limitation.
2
, the increased K
m
value and the decreased
SIase, with considerably improved pH, thermal, and operational
stability. The novel biocatalyst material, a nanostructured crys-
talline, can be easily separated from the reaction medium by natu-
v
2
might have resulted from the steric hin-
ral settling or low-speed centrifugation. SI-EPL-M-TiO
excellent reusability in enzymatic catalysis system over 16 succes-
sive cycles. M-TiO functionalized by EPL has great potential to be
used in other enzyme systems and can be applied as a practical
enzymatic bioreactor in food industries.
2
showed
3
.7. Repeated enzymatic conversion of sucrose to isomaltulose and
2
trehalulose using immobilized enzymes
Strong reusability of immobilized enzymes is quite important,
because it is a crucial quality to be used as industrial catalysts
Acknowledgments
(
Goradia, Cooney, Hodnett, & Magner, 2006). A repeated batch cat-
alytic reaction was designed to determine the practical reusability
of SI-M-TiO and SI-EPL-M-TiO under optimal conditions (Fig. 4).
The two kinds of immobilized SIase could catalyze 50 g/L of
sucrose into isomaltulose and trehalulose. The SI-M-TiO shows a
This work was supported by the National Basic Research
Program of China (2013CB733603), the National High Technology
Research and Development Program of China (2012AA021503),
the National Nature Science Foundation of China (31371732),
and the Program for Changjiang Scholars and Innovative
Research Team in University (IRT1066).
2
2
2
general operational stability, the sucrose conversion rate is
decreased by less than 90% after 5 batches. The sucrose conversion
rate of SI-M-TiO
life is about 55 h. Inversely, the sucrose conversion rate remains at
approximately 95% by SI-EPL-M-TiO after 16 conversion cycles.
2
is 65% at the end of the 8th recycle, and the half-
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