Biochemical Engineering Journal 46 (2009) 186192

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Biochemical Engineering Journal

j o u r n a l h o m e p a g e : w w w. e l s e v i e r.c o m / l o c a t e / b e j

Immobilized transglucosidase in biomimetic polymerinorganic hybrid capsules

for efcient conversion of maltose to isomaltooligosaccharides
Lei Zhang, Yanjun Jiang, Zhongyi Jiang

, Xiaohui Sun, Jiafu Shi, Wei Cheng, Qianyun Sun

Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, 92 Weijin
Road, Nankai District, Tianjin 300072, PR China

a r t i c l e

i n f o

Article history:
Received 21 January 2009
Received in revised form 6 May 2009
Accepted 8 May 2009

Keywords:
Enzyme
Immobilized
Biocatalyst preparation
Kinetics
Biomimetic hybrid capsule
Isomaltooligosaccharide

a b s t r a c t
Isomaltooligosaccharides (IMOs) are relatively new functional food ingredients which have great
poten- tial to improve the quality of many foods due to their low calories, no cariogenicity and
safety for diabetics. To convert maltose to IMOs efciently, -transglucosidase was immobilized in
a kind of alginatechitosancalcium phosphate hybrid capsules (AlgChiCaP), which were
prepared through a facile bio-inspired mineralization process. The surface morphology of Alg
ChiCaP capsule and alginatechitosan capsule (AlgChi) was characterized by scanning electron
microscopy (SEM). Due to the presence of inorganic shell, immobilization efciency of transglucosidase
in AlgChiCaP capsules was higher than that in AlgChi capsules. The optimal temperature (60 C)
and pH (6.0) value for enzymatic conversion catalyzed by transglucosidase immobilized AlgChiCaP
capsules were identical to those catalyzed by free transglucosidase. As compared to the free enzyme,
transglucosidase in AlgChiCaP capsules exhibited signicantly higher recycling stability and storage
stability in a broader temperature and pH range.
2009 Elsevier B.V. All rights reserved.

1. Introduction
In recent years, considerable R&D efforts have been devoted to
oligosaccharide engineering with the focus on exploring the
func- tion of oligosaccharides for mammalian metabolic process
[13]. Oligosaccharides are
relatively new functional food
ingredients which have great potential to improve the quality of
many foods. Various oligosaccharides
[4],
including
isomaltooligosaccharides (IMOs) [5], fructooligosaccharide [6] and
soybean oligosaccharides [7], have been widely used for food or
feed additives [8] and scaffold [9,10] due to their potential
advantages such as bidus- stimulating activity [11], low caloric
value [12] and low cariogenic properties [13]
etc.
IMOs,
comparing with other oligosaccharides, have received peculiar
attention since they are very stable in acid solution, relatively low
in price and have extensive sources [4]. Commercially available
IMO is dened as saccharides that have 40%
-(1-6) glucosidic linkages among the total linkages. IMOs with
the degrees of polymerization (DP) ranging from 2 to 6 are produced from corn starch by serial reactions of starch with
-amylase and
-amylase and transglucosidase [14].
-transglucosidase from Aspergillus niger [15,16] catalyzes the
transglucosylation to the 6- OH of the accepting glucose unit and
yielded the oligosaccharides

Corresponding author. Tel.: +86 22 2350 0086; fax: +86 22 2350 0086.
E-mail address: zhyjiang@tju.edu.cn (Z. Jiang).
1369-703X/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2009.05.008

with an a-D-(1-6) linkage including isomaltose, panose and

isomal- totriose. Compared to free enzyme, immobilized enzyme
improved the operational stability and reusability, which could
better meet requirement for industrial application. However, as
far as we were concerned, immobilized transglucosidase has not
yet been utilized for enzymatic production of IMOs.
The polymerinorganic hybrid carrier have found increased
application in enzyme immobilization due to its moderate
hydrophilicity, controllable transport characteristics, and good
physicochemical stability [17,18], which may create a benign
microenvironment for enzymes [19]. At present, two kinds of congurations can be found for polymerinorganic hybrid carriers:
the mixedmatrix conguration [20] and the coreshell conguration [21]. Compared with the mixedmatrix conguration, the
coreshell conguration could create a more nature-like environment for the immobilized enzyme. Chitosan and alginate are
natural cationic or anionic polysaccharides, respectively, which
have been successfully utilized in biomacromolecule encapsulation [2225]. Furthermore, chitosan has been regarded as an
efcient structure-directing agent for inorganic minerals [26]. Calcium phosphate, a principal component of hard tissues such as
bone and tooth enamel [27,28], is of superior biocompatibility,
insolubil- ity
and mechanical stability, which have been
demonstrated to be suitable for the enzyme immobilization [29].
In this work, alginatechitosancalcium phosphate hybrid
capsules (AlgChiCaP) were employed to immobilize transglucosidase for efcient conversion of maltose to IMOs. The capsules

18

L. Zhang
L. Zhang
et al.
et /al.
Biochemical
/ Biochemical
Engineering
Engineering
Journal
Journal
46 (2009)
46 (2009)
186
186
192192

were produced by a one-step method in which the deposition of

a semi-permeable alginatechitosan lm around droplets of
sodium alginate was coupled with in situ precipitation of
calcium phos- phate. Ca2+
cross-linked alginate containing
transglucosidase was rst coated with chitosan and then coated
with calcium phos- phate to format AlgChiCaP capsules with
enhanced mechanical strength and decreased enzyme leakage.
The
optimum catalytic condition, kinetic parameters, the
recycling
and
storage
stability
of
the
immobilized
transglucosidase were studied extensively.

18

2.4. Immobilization of transglucosidase

Sodium alginate [2.0% (w/v)] dissolved in HPO4 2 solution containing 3 mg/ml transglucosidase was prepared. For one batch,
1 ml of the mixture was added dropwise into a gently stirring
Ca2+ -containing chitosan solution [1.0% (w/v)] with a syringe. The
capsules formed were left immersed in the chitosan solution for
30 min, before being ltered off and rinsed in an excess of
distilled water.
2.5. Activity assays of transglucosidase

2. Materials and methods

2.1.
Materials
Transglucosidase
(EC3.2.1.20, from
A.
niger)
was
obtained from megazyme. Chitosan (deacetylation degree 7585%,
viscosity
20200 cps) were obtained from SigmaAldrich. Sodium alginate
(average molecular weight, 6.27 105 ) was obtained from
Shang- hai Tianlian, China. Maltose was obtained from Guangfu,
China. All other reagents used were of analytical grade and were
used without further purication.

2.2. Preparation of polymerinorganic hybrid capsule

Sodium alginate and disodium hydrogen phosphate (100
mM) solution were mixed to a nal concentration of 2.0% (w/v).
Chi- tosan and calcium chloride solution (100 mM) were mixed to
a nal concentration of 1.0% (w/v). Alginatechitosancalcium
phosphate hybrid capsules (AlgChiCaP) capsules were prepared
by the drop- wise addition of alginate solution into a gently
stirring chitosan solution, using a 5 ml syringe with a 0.9 mm
diameter needle as shown in Fig. 1. The capsules formed were left
in the chitosan solu- tion for 30 min, before being ltered off
and rinsed in an excess of distilled water. Alginatechitosan
capsules (AlgChi) capsules were prepared following the same
procedure, with the exception of alginate mixing in distilled
water instead of HPO4 2 ions. All procedures were carried out
at room temperature and pH 7.0.
2.3. Characterizations of capsules
Intact capsules were observed with Zoom Stereo Microscope
(Olympus SZ2-ILST). The morphology of capsule surface was
observed with scanning electron microscopy (SEM, XL30,
PHILIPS, Holand) using an accelerating voltage of 20 kV; before
analyzing, the capsule was rst freeze-dried and gold coated.

The activities of free and immobilized transglucosidase were

determined by the transglycosylation of maltose. The free or
immo- bilized transglucosidase and 5 ml, 100 mg/ml maltose
solution were mixed and the system was incubated in a water
bath with con- stant shaking at different temperature for 10 min.
The reaction was stopped by adding two times volume of
acetonitrile reagent. Incu- bation was performed in a boiling water
bath for 5 min. An enzyme activity unit (U) was dened as the
amount of enzyme liberating
1 mg maltose per minute under the assay conditions. Each result
was an average of four or ve separate experiments.
Maltose was quantied using high performance liquid chromatography (HPLC) operating on an analytical column (Tsk-gel
Amide80, 5 m, 4.6 mm id 250 mm). After 10 min reaction, 60
l of the digested maltose solution was diluted with 140 l of acetonitrile. HPLC was used to analyse the samples. For the elution
conditions; the mobile phase was 70% acetonitrile and 30% water.
Temperature was kept constant at 50 C, with a ow rate of
0.8 ml/min and an injection volume of 20 l. The detector was
a Knauer Differential-Refractometer. In each set of experiment, a
standard curve was plotted with maltose solutions of different
con- centrations.
2.6. Immobilization efciency
The immobilization efciency of capsules was determined using
the following equation.
immobilization efciency (%)
[transglucosidase]solution Vsolution
100
(1)
[transglucosidase] droplet Vdroplet
where [transglucosidase]solution and [transglucosidase]droplet are
the concentration of transglucosidase in the nal solution and in
the original liquid droplet, Vsolution and Vdroplet represent the
volume of the solution and liquid drop, respectively. The
transglucosidase concentration was determined by the enzyme
activity.
=
100

Fig. 1. Schematic illustration of the enzyme immobilization process.

2.7. Stirring speed

2.11. Recycling stability and storage stability

An experiment was done to choose the optimum stirring

speed. The optimum stirring speed of reaction were determined
by immersing transglucosidase-containing capsules into 5 ml,
100 mg/ml of maltose at 50 C and pH 6.0 for 30 min, and the concentration of transglucosidase changes were monitored at
specied time intervals.

The recycling stabilities of the immobilized transglucosidase

were evaluated by measuring the enzyme activity in each successive reaction cycle, and were expressed by recycling efciency.
The immobilized transglucosidase were allowed to take effect in
mal- tose solution with pH 6 and 50 C (optimum). The
immobilized transglucosidase was then ltered off, rinsed with
distilled water, and then, used in the next reaction cycle. The
process was repeated for 15 times.
The activity found for each repetition was compared with the
initial activity assuming it possessed 100% activity.

2.8. Leakage of transglucosidase from AlgChi and AlgChiCaP

capsules
The leakage characteristics of capsules were determined by
immersing capsules into 5 ml of deionized water at room
temper- ature and 600 rpm/min, and the concentration of
transglucosidase changes were monitored at specied time
intervals. The
leakage was calculated using the following
equation:
leakage
(%)
=

[transglucosidase]solution Vsolution 100

[transglucosidase] droplet Vdroplet

(2)

2.9. Optimum conditions for enzyme activity

The optimum temperature of the free
glucosidase was evaluated by adding
maltose solution for 10 min under
condition. Temperatures of 5, 25, 37, 50, 60, and 70 C
=

recycling efciency (%)

=

enzyme activity on the storing for nth day

100
initial enzyme activity on the 1st cycle

(6)

Many batches of the free and immobilized transglucosidase

were prepared and stored in glass vials at 4 C to determine the
storage efciency. On the 1st, 2nd, 3rd, 5th, 7th, 10th, 13th, 15th,
17th, 20th, 23rd, 27th and 30th day, 1 ml of each type of the free
and immobilized transglucosidase was added to 5 ml of maltose
solution at pH 6 and under temperature of 50 C.

and immobilized transthe enzyme into the

different temperature

storage efciency (%)

enzyme activity after storing for nth day

were used for the experiment.

initial enzyme activity

100

(7)

The activity found at each temperature was compared with

the activity at optimum temperature assuming it possessed
100% activity.
enzyme activity at the specic temperature
100
enzyme activity at optimum temperature
activity (%)
(3)
=

3. Results and discussion

The optimum pH of the free and immobilized

transglucosidase was evaluated by adding the enzyme into the
maltose solution for
10 min. Maltose solutions with pH of 2, 3, 4, 5, 6, 7, 8 and 9 were
used. The activity found at each pH was compared with the
activity at optimum pH assuming it possessed 100% activity.

As shown in Fig. 3, AlgChiCaP capsules were prepared by

the dropwise addition of phosphate-containing sodium alginate
solution into calcium-containing chitosan solution. Because of
interfacial complexation of the oppositely charged polysaccharide,
a thin chitosan lm formed around the alginate droplets spontaneously. Counter-diffusion of the oppositely charged ions across
the polysaccharide interface results in the in situ precipitation of
cal- cium phosphate. The capsules became much stiffer due to
further
cross-linking
between
alginate and Ca2+ ions and the deposition of
calcium phosphate
[30].

activity(%) =

enzyme activity at the specic pH

100
enzyme activity at optimum pH
(4)

The micrographs of AlgChi and AlgChiCaP capsules were

2.10. Determination of Km and Vmax values

Activities of the free and immobilized transglucosidase were
determined by using the classical MichaelisMenten kinetics. In
the graphical evaluation of MichaelisMenten constants and
maximum activities, LineweaverBurk plots obtained by plotting
experimen- tal values were used.
1 = Km
V
Vmax
Vmax

1+
.
[S]

3.1. Characterization of the capsules

(5)

V and [S] are the initial reactive rate and initial substrate
concentra- tion, respectively. Vmax is the maximum activity
attained at innite initial substrate concentration and Km is the
MichaelisMenten constant.
To determine Vmax and the Km , the activity assay was applied
for different maltose concentrations (10, 12.5, 16.7, 25, 50
and

shown in Fig. 2a and b, respectively. It could be observed that the

AlgChi capsules were more jelly-like whereas the AlgChiCaP
capsules were noticeably harder. Fig. 2c and d showed that the
AlgChiCaP capsule had smooth and intact surface structure, in
comparison, the AlgChi capsule had a wrinkled surface structure, which was ascribed to the polymer shrunk during the
freeze drying process. As shown in Fig. 2e and f, the internal
surface of the capsule was Ca2+ -alginate hydrogel network and
the external
surface of the capsule was relatively smooth calcium phosphate
100 mM). Activities of free and immobilized transglucosidase
were all determined at the optimum conditions. The catalytic
efcien- cies of both free and immobilized transglucosidase were
calculated accordingly.

layer.
3.2. Immobilization efciency
The immobilization efciency could be assessed by measuring the transglucosidase concentration in the solution during

the immobilization. The immobilization efciency was 92.6% for

AlgChiCaP capsules, whereas it was 70.5% for AlgChi capsules.
Due to existence of the external inorganic layer, the leakage of
transglucosidase was considerably reduced during the capsules
for- mation process.

Fig. 2. Optical micrographs of (a) AlgChi capsules and (b) AlgChiCaP capsules; SEM image of (c) AlgChi capsules, (d) AlgChiCaP capsules, (e) AlgChiCaP capsules
interior and (f) AlgChiCaP capsules exterior.

3.3. Stirring speed

When stirring speed was greater than 400 rpm/min, it had
trivial inuence on reaction rate because the internal diffusion
controlled the whole diffusion process as shown in Fig. 3. When
the stirring

speed was less than 400 rpm/min, the reaction rate reached
the highest, which was due to that the external diffusion
constituted the control step for the whole diffusion process. In the
following exper- iment, the stirring speed used was 400 rpm/min
unless otherwise noted.
3.4. Leakage of transglucosidase
To assess the leakage transglucosidase from the AlgChi and
AlgChiCaP capsules, both capsules were dip in deionized water
at 1000 rpm/min. As seen in Fig. 4, extended time studies
indicated that over 80% of the immobilized transglucosidase
were leaked from the AlgChi capsules to the surrounding
solution within a period of 5 h. In contrast, capsules prepared
with calcium phos- phate retained approximately 65% of the
trapped transglucosidase after 7 h, indicating that in situ
precipitation of calcium phosphate process was successful in
signicantly reducing the enzyme leakage from the capsules.
3.5. Thermal and pH stabilities of immobilized transglucosidase

Fig. 3. Effects of stirring speed (50 C, pH 6.0, reaction time 30 min) on the
maltose conversion ratio catalyzed by immobilized transglucosidases.

The activity of free and immobilized transglucosidase was

assayed at various temperatures (570 C) as shown in Fig. 5a.
The results showed that optimum reaction temperature (50 C)
was not affected by immobilization. The transglucosidase
immobilized

Fig. 4. The leakage of transglucosidase from AlgChi capsules and AlgChiCaP

capsules.

in AlgChiCaP capsules displayed the broadest temperature

pro- le, indicating that immobilization could render enzyme
with a more benign environment which protected enzyme
from heat- induced denaturation and allowed the enzyme to
become less
temperature-depending. At 70 C, approximately 88% and 66% of
the initial activity was lost for the free enzyme and immobilized enzyme, respectively. It is well established that thermal
inactivation starts with the unfolding of the protein molecule
which is followed by irreversible changes due to aggregation and formation of scrambled structures which takes place

Fig. 5. (a) Effects of temperature (pH 6.0, reaction time 10 min) on the activity
of free and immobilized transglucosidases. (b) Effects of pH value (50 C, reaction
time

more in soluble form as compared to the immobilized state

[31,32].
As shown in Fig. 5(b), the effect of pH on the activity of
free and immobilized transglucosidase was studied (from pH 2.0
to 9.0.) and found that both free and immobilized transglucosidase were sensitive to the pH changes. The highest activity for
immobilized transglucosidase was achieved at pH 6.0, completely
consistent with that of free transglucosidase, which further supported that the conformation of the transglucosidase was well
preserved after immobilization. Transglucosidase immobilized in
AlgChiCaP capsules retained 77% of its maximum activity at pH
2.0 and 53% at pH 9.0, whereas, free transglucosidase retained
only 33% of its maximum activity at pH 2.0 and 29% at pH
9.0, respectively. Such changes are generally analyzed as a result
of immobilization, which greatly helped in the stabilization of
enzyme at a wider pH range [31,33]. Additionally, it was noticed
that enzyme-containing capsules preserved higher activity in acid
condition than in alkaline condition. The strong resistance of
immo- bilized transglucosidase against the acidic medium was
tentatively explained by the buffering effect of the alginate
capsule. According to the previous report, the pKa of alginate
ranges between 3.4 and
4.4, which was attributed to the ionization of the unbound
carboxyl
group (not bound to Ca2+ -ion) [34,35]. In acidic medium, the
negatively charged alginate core could attract and consume the + ions,
H
10 min) on the
transglucosidases.

activity

of

free

and

immobilized

assisting in preventing H+ ion from diffusing into and contacting

with the enzymes [36].

was 1.17 and 1.18 U/mg, respectively. Lower specic activity

of immobilized transglucosidase might be due to the additional
dif- fusion resistance rather than enzyme denaturation.

3.6. Free and immobilized enzyme activity under optimal

conditions
Under optimum condition (50 C and pH 6.0), the enzyme
activ- ity of free and immobilized transglucosidase in AlgChi
capsules and AlgChiCaP capsules was investigated. As shown in
Fig. 6, the bioconversion process was monitored by measuring the
conversion of maltose with the lapse of time. The reaction rate
and the nal conversion rate using immobilized transglucosidase
were slightly lower than that of free transglucosidase. The
equilibrium conver- sion using free transglucosidase was
obtained at 95.31% in 13 h, while that in the case of immobilized
transglucosidase immobilized in AlgChi capsules was 96.99% in
13.83 h and in AlgChiCaP cap- sules was 97.4% in 13.98 h. The
enzyme activity unit was dened as
the amount of
transglucosidase needed to convert 1.0 mg of maltose/min at 50

C, pH 6.0. The specic activity of free trans- glucosidase was

1.22
U/mg,
while
specic
activity
of
immobilized
transglucosidase in AlgChi capsules and in AlgChiCaP capsules
3.7. Kinetic studies
The MichaelisMenten kinetics of free and immobilized transglucosidase was studied and the corresponding Michaelis constant
(Km ) and maximum reaction rate (Vmax) were calculated from
LineweaverBurk plots.
The value of the maximum reaction rate (Vmax ) and the
Michaelis constant (Km ) evaluated from LineweaverBurk plots
were shown. Both the Km and the Vmax values were changed by
the immobiliza- tion processes. The
Vmax value of the
transglucosidase in AlgChi capsules (1.025 U/mg) and AlgChi
CaP capsules (1.165 U/mg) was found to be lower than that of
free transglucosidase (1.239 U/mg). The Km value of the
transglucosidase in AlgChi (16.015 mM) and AlgChiCaP (22.37
mM) capsules were found to be higher than that of the free
transglucosidase (9.094 mM). The decreased Vmax value of the
immobilized transglucosidase could be due to the additional
diffusion limitation to the substrate (maltose) and the product
(IMOs) caused by the carrier, which induced the low accessibility of the substrate to the active sites of the transglucosidase
and consequently resulted in a lower possibility of enzyme
substrate complex formation. The Km value was known as the
afnity of the enzymes toward substrates and the lower values
of Km meant the higher afnity between enzymes and substrates.
[37] The increase in Km after immobilization indicated a weaker
binding between the maltose molecules and the immobilized
transglucosidase.
3.8.
Recycling
stability

stability

and

storage

Enzymes were very sensitive to environmental conditions

and might lose their activities quite easily. Thus, it was
meaningful to characterize their recycling stability and storage
stability for industrial application. To determine the recycling
stability of the immobilized enzyme, the activity of
transglucosidase in capsules was measured sequentially 15
times at the optimum condition (50 C and pH 6.0). After one
cycle, capsules containing enzyme were removed from the
reaction medium and washed twice with distilled water.
As shown in Fig. 7, after the 7th reaction cycles, the
transglucosi- dase in AlgChi capsule lost half of its initial activity,
whereas in the AlgChiCaP capsule, after the 15th cycles, it still
retained 65% of its initial activity. The difference in recycling
stability was due to the leakage of transglucosidase during the
multiple soaking, sep- aration, and washing processes employed
in the reaction cycles. Whereas, for the transglucosidase
immobilized in AlgChiCaP capsule, transglucosidase leakage
was prevented during recycling process and the recycling

Fig. 6. Maltose conversion with reaction time (50 C, pH 6.0).

stability was increased substantially. The pore size

of the
inorganic layer should meet two requirements: it must be big
enough for the substrates and products to pass through freely but
small enough to effectively prevent the enzyme from leaking.
Since BET analysis showed the average pore diameter of the Alg
ChiCaP capsule was slightly bigger than 3 nm [38], it could be
concluded that AlgChiCaP capsule was effective as it allowed
for the substrates and products (0.6 nm) to pass through, and
mean- while prevented transglucosidase which was larger than 3
nm from leaking from capsules.
In addition, the free and immobilized enzymes were stored
without any buffer at 4 C and their activities were tested for 30
days. The
storage stability of free and immobilized
transglucosidase
was shown. Taking the initial activity level to be 100%, the relative
activity
of
the
free
transglucosidase,
transglucosidase
immobilized in AlgChi and AlgChiCaP capsules was decreased
to 38%, 78%, and 85% after 30 days, respectively. The little
differences in the storage stability of the AlgChi and AlgChi
CaP capsule indicated that the calcium phosphate played trivial
role in improving storage stability. It was reasonably believed
that the immobilized trans- glucosidase would exhibit a distinct
advantage over free enzyme in

long-time storage, owing to crowded microenvironment created

by the biomimetic alginate capsule, which closely imitated the
effects of crowding and connement in a living cell. Alginate and
trans- glucosidase (pI = 5.1) both bear net negative charges in a
neutral pH environment. Therefore, the conformational transition
of trans- glucosidase from a folded to an unfolded state was
substantially inhibited by the electrostatic repulsion between
transglucosidase and alginate molecules. Additionally,
the
biocompatible alginate helped the enzymes avoid the
unfavorable attack possibly arising from the outside storage
environment effectively.
4. Conclusions

Fig. 7. (a) Recycling stability of immobilized transglucosidases in AlgChi

capsules and AlgChiCaP capsules. (b) Storage stability of free and immobilized
transglu- cosidases.

A facile method for preparing alginatechitosancalcium

phosphate hybrid capsules (AlgChiCaP) as efcient transglucosidase immobilization carrier was proposed. The biocompatible
alginate-core accommodated the suitable microenvironment for
transglucosidase, the outer calcium phosphate shell of capsule
ensured the facile accessibility of immobilized transglucosidase for
substrates and prevent transglucosidase from leaking out effectively. Owing to the synergy effect of hydrophilic polymers and
mechanically stable calcium phosphate, the immobilized transglucosidase displayed higher thermal stability and pH stability than
that in the free form and retained more than 60% initial activity
after
15
repeated
cycles.
Additionally,
compared
to
free
transglucosidase, the immobilized transglucosidase exhibited
improved storage sta- bility. The facile immobilization process
and the enhanced stability set an encouraging example for
converting natural compounds into high value-added functional
products.

Acknowledgements
The authors thank the nancial support from the National
High- Tech
Research
and Development Plan
(no.
2007AA10Z305), the Cross-Century Talent Raising Program of
Ministry of Education of China, the program for Changjiang
Scholars Innovative Research Team in University (PCSIRT) and
the Programme of
Introducing Talents of Discipline to
Universities (no. B06006).
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