Enzymatic transformation of 2-O-α-d-glucopyranosyl-L-ascorbic acid (AA-2G) by immobilized α-cyclodextrin glucanotransferase from recombinant Escherichia coli

Article abstract of DOI:10.1016/j.molcatb.2010.11.009

This work aims to produce 2-O-α-d-glucopyranosyl-L-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin with immobilized α-cyclodextrin glucanotransferase (α-CGTase) from recombinant Escherichia coli. Molecular sieve (SBA-15) was used as an adsorbent, and sodium alginate was used as a carrier, and glutaraldehyde (GA) was used as a cross-linker. The effects of several key variables on α-CGTase immobilization were examined, and optimal immobilization conditions were determined as the following: glutaraldehyde (GA, cross-linker) 0.01% (v/v), SBA-15 (adsorbent) 2 g/L, CaCl₂ 3 g/L, sodium alginate 20 g/L, adsorption time 3 h, and immobilization time 1 h. In comparison with free α-CGTase, immobilized α-CGTase had a similar optimal pH (5.5) and a higher optimal temperature (45 °C). The continuous production of AA-2G from ascorbic acid and β-cyclodextrin in the presence of immobilized α-CGTase was carried out, and the highest AA-2G production reached 21 g/L, which was 2-fold of that with free α-CGTase. The immobilization procedure developed here was efficient for α-CGTase immobilization, which was proved to be a prospective approach for the enzymatic production of AA-2G.

Full text of DOI:10.1016/j.molcatb.2010.11.009

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 223–229
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enzymatic transformation of 2-O-␣-d-glucopyranosyl-l-ascorbic acid (AA-2G)
by immobilized ␣-cyclodextrin glucanotransferase from recombinant
Escherichia coli
Zichen Zhanga,b, Jianghua Lia,b,1, Long Liua,b, Jun Sun , Zhaozhe Huaa,b, Guocheng Dua,∗, Jian Chend,∗∗
c
a
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Lihu Road 1800, Wuxi 214122, China
School of Biotechnology, Jiangnan University, Wuxi 214122, China
Institute of Information Technology, Jiangnan University, Wuxi 214122, China
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 August 2010
Received in revised form
This work aims to produce 2-O-␣-d-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and
␤
-cyclodextrin with immobilized ␣-cyclodextrin glucanotransferase (␣-CGTase) from recombinant
Escherichia coli. Molecular sieve (SBA-15) was used as an adsorbent, and sodium alginate was used as
a carrier, and glutaraldehyde (GA) was used as a cross-linker. The effects of several key variables on ␣-
CGTase immobilization were examined, and optimal immobilization conditions were determined as the
following: glutaraldehyde (GA, cross-linker) 0.01% (v/v), SBA-15 (adsorbent) 2 g/L, CaCl2 3 g/L, sodium
alginate 20 g/L, adsorption time 3 h, and immobilization time 1 h. In comparison with free ␣-CGTase,
1
8 November 2010
Accepted 18 November 2010
Available online 26 November 2010
Keywords:
l-Ascorbic acid
◦
immobilized ␣-CGTase had a similar optimal pH (5.5) and a higher optimal temperature (45 C). The
continuous production of AA-2G from ascorbic acid and ␤-cyclodextrin in the presence of immobilized
␣-CGTase was carried out, and the highest AA-2G production reached 21 g/L, which was 2-fold of that with
free ␣-CGTase. The immobilization procedure developed here was efficient for ␣-CGTase immobilization,
which was proved to be a prospective approach for the enzymatic production of AA-2G.
␣
2
-Cyclodextrin glucanotransferase
-O-␣-d-glucopyranosyl-l-ascorbic acid
Immobilization
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
like AA 2-O-phosphate or AA 2-O-sulfate in terms of the reaction
specificity and efficiency in large-scale production [7]. In addi-
tion to being stable in vitro and highly active in vivo, AA-2G also
exhibits lipophilicity and may be used as components in cosmetics
[8–10].
Cyclodextrin glucanotransferase (EC 2.4.1.19, CGTase) is one of
the important amylolytic enzymes and finds wide applications in
the production of cyclodextrins (CDs) and ␣-1,4-linked cyclicol-
igosaccharides [1]. CGTase can be used for the production of CDs via
intra-molecular transglycosylation reaction, and also can be used
for the production of functional substances like glycosylated ascor-
bic acid (AA) via intermolecular transglycosylation [2–4], in which
the glycosyl residue is transferred from ␣-1,4-glucan or cyclodex-
trin to a suitable acceptor such as AA or sucrose [5].
The enzymatic production of AA-2G is more applicable than
chemical synthesis of the other AA derivatives due to the sim-
ple reaction steps, high regiospecificity and low production cost.
The enzymatic transformation of AA-2G via the transglycosylation
with CGTase is attracting an increasing interest. Markosyan et al.
produced AA-2G with CGTase from mesophilic, thermophilic, and
halophilic bacteria and maltase from the yeast Saccharomyces cere-
visiae [11]. Jun et al. produced AA-2G via transglycosylation by
CGTase from Paenibacillus sp. [12]. Currently, in most studies the
CGTase used for the transformation of AA-2G is free, and there are
few reports concerning the AA-2G production with the immobi-
lized CGTase, though the latter has many advantages. Prousoontorn
and Pantatan investigated the production of AA-2G with the immo-
bilized CGTase from Paenibacillus sp. [2]. In their work, a covalent
coupling method was used and the performance of the immobi-
lized CGTase was evaluated. However, the immobilized CGTase
cannot be used for the large scale production of AA-2G due to the
low CGTase activity and AA-2G yield. Therefore, how to achieve
Muto et al. found a type of glucosylated AA, namely, 2-
O-␣-d-glucopyranosyl-l-ascorbic acid (AA-2G), which can be
enzymatically synthesized via transglucosylation with CGTase
[
5,6]. AA-2G is extremely stable and nonreducible, and is consid-
ered to be superior to other chemically synthesized AA derivatives
∗
∗
Corresponding author. Tel.: +86 510 85918309; fax: +86 510 85918309.
Corresponding author. Tel.: +86 510 85913661; fax: +86 510 85910799.
E-mail addresses: gcdu@jiangnan.edu.cn (G. Du), jchen@jiangnan.edu.cn
∗
(
J. Chen).
1
Shared with the first author.
1
381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.11.009

2
24
Z. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 223–229
the efficient CGTase immobilization is important for the enzymatic
production of AA-2G.
as the amount of enzyme that was able to produce 1 ␮mol of
␣-cyclodextrin per min under the above conditions.
CGTase has been immobilized on different supports by covalent
binding, adsorption, entrapment or cross-linking [13–17]. A few
reports have described the immobilization of CGTase covalently to
supports, such as Eupergit C and glyoxyl-agarose [18–20], or by
entrapment in sodium alginate beads [21,22], or by exchange with
ionic bond [23].
2.2.2. Glucoamylase activity assay
One unit of glucoamylase activity is defined as the amount of
enzyme which produces 1.0 mmol glucose from dextrin in 1.0 min
◦
at 50 C at pH 5.5 [30].
Sodium alginate is a suitable carrier for entrapment immobi-
lization of enzymes for industrial applications [24]. Glutaraldehyde
activation of supports is one of the most popular techniques to
immobilize enzymes. The glutaraldehyde is used to activate sup-
ports by physical adsorption and covalent binding, immobilizing
the enzyme on a glutaraldehyde pre-activated support [25–27].
2.3. Preparation of enzyme solution
The recombinant plasmid cgt/pET-20b(+), in which the cgt gene
encoding the mature ␣-CGTase from Paenibacillus macerans strain
JFB05-01(CCTCC M203062) was placed downstream of a DNA
sequence coding pelB signal peptide, and Escherichia coli BL21(DE3)
harboring plasmid cgt/pET-20b(+) were constructed previously
[31].
The culture conditions for the extracellular production of
CGTase from recombinant E. coli BL21 (DE3) in shaking flask were
(g/L): glucose 8, lactose 0.5, peptone 12, yeast extract 24, K HPO4
SBA-15 is porous silicates with huge surface areas (normally
2
≥
500 m /g), large pore sizes (2 nm ≤ size ≤ 20 nm) and ordered
arrays of cylindrical mesopores with very regular pore morphol-
ogy. The large surface areas of these solids increase the probability
that a reactant molecule in solution will come into contact with
the catalyst surface and react. The large pore size and ordered pore
morphology allow one to be sure that the reactant molecules are
small enough to diffuse into the pores [28]. Chemical modifica-
tions of amino acids reduce any conformational change involved
in enzyme inactivation and increase the enzyme stability.
2
16.4, KH PO4 2.3, and CaCl2 0.28. The initial pH was 7.0 and the
2
◦
culture temperature was maintained at 25 C. The culture broth
◦
was centrifuged at 12,000 × g at 4 C for 5 min and the collected
supernatant was hyperfiltrated.
In this work, the ␣-CGTase was immobilized with SBA-15 as
an adsorbent, glutaraldehyde as a cross-linking agent, and sodium
alginate as a carrier. Characterization of the immobilized enzyme
for catalytic properties in comparison with the free enzyme was
also investigated. The immobilized ␣-CGTase was used to repeat
the production of AA-2G from ascorbic acid and ␤-cyclodextrin. It
should be noted that here the developed ␣-CGTase immobilization
is not a novel immobilization approach, but reaches a significant
enhancement of AA-2G production via the combination of exist-
ing materials and technologies. This work may be helpful for the
industrial production of AA-2G with the immobilized ␣-CGTase.
2.4. Enzyme immobilization methods
2.4.1. Embedding of ˛-CGTase
Thoroughly mixed solution of 10 mL (160 U/mL) enzyme and
0.2 g sodium alginate was placed into 4 C refrigerator overnight
for degasification. The drops of the solution were added into CaC12
(3%) for 2 h hardening. The collected capsules were washed with
distilled water until there was no protein precipitation. The washed
◦
◦
capsules were stored in glycerol at 4 C until subsequent use.
2.4.2. Crosslinking-embedding of ˛-CGTase
Ten milliliters (160 U/mL) of enzyme solution and 100 ␮L of glu-
taraldehyde (25%) were mixed completely and then cross-linked
for 4 h. The mixture was added with 0.2 g sodium alginate, and
2
. Materials and methods
◦
then was placed in 4 Crefrigerator overnight for degasification. The
2.1. Materials
drops of the solution were added into CaC1 (3%) for 2 h hardening.
2
The collected capsules were washed with distilled water until there
was no protein precipitation, and then the washed capsules were
stored in glycerine at 4 C until subsequent use.
Sodium alginate, glutaraldehyde (25% aqueous solution, GA),
CaCl , ␤-cyclodextrin and ascorbic acid were purchased from San-
2
◦
gon (Shanghai, China). Molecular sieve (SBA-15) was purchased
from Dragon Technology Co., Ltd. (Liaoning, China). AA-2G was
purchased from Wako Pure Chemical (Wako, Japan). All other
chemicals and reagents were of analytical grade.
2.4.3. Embedding-crosslinking of ˛-CGTase
The capsules collected via embedding method were washed
with distilled water until there was no protein precipitation, and
were further cross-linked for 4 h in 30 mL of glutaraldehyde solu-
tion (0.2%). The treated capsules were preserved in glycerol.
2
2
.2. Activity measurements
.2.1. ˛-CGTase activity assay
2.4.4. Adsorption-crosslinking-embedding of ˛-CGTase
The ␣-recombinant CGTase was assayed as the dextrinogenic
The mixture consisting of 10 mL (160 U/mL) of enzyme solu-
tion and 0.01–0.1 g of molecular sieve was placed into a shaker at
16 C and 150 rpm for 1–10 h adsorption, and then 100 ␮L of 25%
activity by methyl orange method described in the literature [29].
The ␣-cyclodextrin forming activity was determined by the methyl
orange method as described previously with some modifications.
The culture or cytoplasmic supernatant (0.1 mL) (appropriately
diluted in 50 mM sodium phosphate buffer) was incubated with
◦
glutaraldehyde was added for 4 h crosslinking. The solution was
◦
treated with 0.2 g sodium alginate and placed in 4 C refrigerator
overnight for degasification. The drops of the solution were added
0
6
.9 mL of 3% (w/v) soluble starch in 50 mM phosphate buffer (pH
.0) at 40 C for 10 min. The reaction was terminated by the addi-
into CaC1 of 2–8% for 2 h hardening. The collected capsules were
2
◦
washed with distilled water until there was no protein precipita-
◦
tion of 1.0 M HCl (1.0 mL), and then 1.0 mL of 0.1 mM methyl orange
in 50 mM phosphate buffer (pH 6.0) was added. After the reac-
tion mixture was incubated at 16 C for 20 min, the amount of
tion, and then the washed capsules were stored in glycerine at 4 C
until subsequent use.
◦
␣
-cyclodextrin in the mixture was spectrophotometrically deter-
2.5. Enzymatic AA-2G production
mined by measuring the absorbance at 505 nm. At the chosen
conditions, the molar extinction coefficient of methyl orange was
ε = 2.98 × 10 L/(mol cm). One unit of ␣-CGTase activity was defined
l-Ascorbic acid and ␤-cyclodextrin were dissolved into 100 mM
acetate buffer to yield final concentration of 5% (w/v) and 5%
4

Z. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 223–229
225
(
w/v), respectively. Five milliliters of the immobilized ␣-CGTase
◦
was added to 5 mL of substrates solution and incubated at 37 C for
4 h with gentle orbital shaking (150 rpm) in the dark. Glucoamy-
2
lase (10 U/mL) was added to the reaction medium and incubated
◦
at 65 C and pH 5.5 for 6 h to hydrolyze the AA-2-oligosaccharides
(
AA-2Gs) to AA-2G. The activity of the immobilized ␣-CGTase was
determined by assaying the rate of AA-2G production in the first
2
4 h.
2.6. Properties of immobilized ˛-CGTase
2.6.1. Effect of pH on the immobilized ˛-CGTase activity
The influence of pH on the activity of immobilized ␣-CGTase
was studied by incubating mixture solutions in sodium acetate
buffer with pH ranging from 4.1 to 6.4 and sodium phosphate buffer
with pH ranging from 6.7 to 7.0. Other conditions are the same as
described in Section 2.5.
Fig. 1. Influences of immobilized enzyme and free enzyme on the synthesis of
AA-2G. (1) Embedding of ␣-CGTase; (2) crosslinking-embedding of ␣-CGTase; (3)
embedding-crosslinking of ␣-CGTase; (4) adsorption-crosslinking-embedding of ␣-
CGTase; (5) free ␣-CGTase.
2
.6.2. Influence of temperature on the immobilized ˛-CGTase
activity
The effect of temperature on the activities of free and immobi-
lized ␣-CGTase was investigated by incubating mixture solutions
at temperatures ranging from 30 to 60 C.
◦
3. Results and discussion
Other conditions are the same as described in Section 2.5.
3.1. Optimization of immobilization procedure
2
.6.3. Reusability
The reusability tests were performed at 45 C with a batch opera-
◦
3.1.1. Immobilization methods
In spite of the fact that a large number of techniques and
supports are now available for the immobilization of CGTase
tion mode. Afterincubation, the immobilized enzyme was removed
from the reaction medium, and was washed three times with citrate
buffer (50 mM, pH 6.0) and then used again.
[
[
2,19,32], there is not a universal means of immobilization
33] due to the fact that the choice of the support as well
as the immobilization technique depends on the nature of the
enzyme and the substrate, and its ultimate application. In this
work, AA-2G concentration formed by free ␣-CGTase is 8.3 g/L
(Fig. 1). Four different immobilization methods were developed,
and the immobilized enzyme demonstrated high activity (>50%),
in particular the adsorption-crosslinking-embedding immobiliza-
tion showed the highest free activity of 90%. The high activity
of adsorption-crosslinking-embedding immobilization may be
attributed to the effective adsorption of free enzyme by SBA-15
as well as the stable covalent structure, which was formed by
glutaraldehyde. Compared to the embedding method, crosslinking-
embedding immobilization achieved a better result, indicating
that enzyme crosslinked with glutaraldehyde was favorable for
transformation. The worse was embedded-crosslinking immobi-
lization, and the possible reason was that the gap formed by
embedding was made so dense by glutaraldehyde crosslinking
that it seriously hindered substrates to enter into to the active
site of the enzyme, leading to a decrease in transformation
efficiency.
2
.6.4. Operational stability
Operational stability of the immobilized CGTase was deter-
mined for AA-2G synthesis in a sealed plastic tube. l-Ascorbic acid
and ␤-cyclodextrin were dissolved into 100 mM acetate buffer to
yield final concentration of 5% (w/v) and 5% (w/v), respectively. The
immobilized ␣-CGTase samples (1600 units) was added to 10 mL
of the substrates solution and incubated in the reactor at 45 C and
3
Ten milliliters of free ␣-CGTase (160 U/mL) was added to 10 mL
of the substrates solution and incubated at 38 C for 5 d with gentle
orbital shaking (150 rpm) in the dark. Samples were taken at regular
time intervals (6 h) for the measurement of AA-2G with HPLC. The
relationship between operation time and conversion was studied.
◦
◦
8 C for 5 d with gentle orbital shaking (150 rpm) in the dark.
◦
2
.6.5. Storage stability
Immobilized ␣-CGTase was stored at 4 C in 50 mM of citrate
◦
buffer. The storage stability of the free and immobilized enzyme
was investigated by measuring their remaining activity once every
1
0 days.
3.1.2. The effect of SBA-15 concentration
Table 1 shows that SBA-15 is a molecular sieve with large
aperture (average adsorption pore diameter 6 nm) and high spe-
cific surface area (≥500 m /g), which can enable fast adsorption
2.7. AA-2G analysis
2
AA-2G was measured with HPLC equipped with a SB-Aq column
Table 1
(
4.6 mm × 250 mm). Samples were previously filtered via a 0.45 ␮m
SBA-15 performance indicators.
membrane before injection. The assay conditions were as follows:
the detection wavelength was 238 nm and the mobile phase was
Index
Value
0
.025 M KH PO /H PO (pH 2.0) at a flow rate of 0.5 mL/min. At
2
2
4
3
4
Surface area (m /g)
≥500
≥0.70
3–4
6
3
these conditions, the standard AA-2G was separated reproducibly.
The AA-2G concentration was calculated on the basis of peak area
of standard curve.
Pore volume (cm /g)
Cell wall (nm)
Average adsorption pore diameter (nm)

2
26
Z. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 223–229
Fig. 2. Effects of SBA-15, GA and CaCl2 concentrations on the conversion rate of the immobilized enzyme: (A) SBA-15, (B) GA and (C) CaCl2.
of enzyme into the network holes. The optimal concentration of
SBA-15 was found to be 2 g/L (Fig. 2A).
3.1.6. Calcification time
At the same conditions, changes in calcification time led to var-
ied conversion rate. As shown in Fig. 3B, the optimal immobilization
time is 1 h. Because Ca2+ played a role in protecting enzyme, 1 h of
calcification time could achieve the optimal immobilization, but
with the extension of time, the conversion rate was inhibited.
3.1.3. The optimum glutaraldehyde concentration
As a crosslinking agent, glutaraldehyde contains two alde-
hyde groups, which can form covalent bonds with enzyme,
yielding three-dimensional crosslinked network structure. A
low concentration (0.01%, v/v) of glutaraldehyde could enhance
the catalytic reaction, however, when the glutaraldehyde con-
centration increased to 0.02%, it would damage the struc-
ture of the enzyme so that the conversion rate decreased
3
3
.2. General properties of free and immobilized ˛-CGTase
.2.1. Effect of pH
A comparative study between free and immobilized enzyme
was provided in terms of pH of the medium. The pH is one of
the most important factors influencing not only the dissociation
of protein protons, but also the solution chemistry of the insolu-
ble support [34–36]. Fig. 4 shows the effect of pH on the activity
of free and immobilized ␣-CGTase. The optimum pH value of both
free and immobilized enzyme was pH 5.0–5.5. This is an expected
result, because the supports did not change the protein structure
of immobilized enzyme acutely.
(
Fig. 2B).
3
.1.4. Influence of CaCl concentration
2
2
+
The Ca concentration may affect the gel mesh size, thus affect-
ing the entrance of substrates into the grid and the efficiency of
enzyme. Therefore, different concentrations of CaCl solution were
used to examine its impact on immobilization of CGTase. Fig. 2C
shows that the concentration of CaC1 has little effect on the con-
2
2
version rate.
3.2.2. Influence of temperature
Fig. 5 shows the influence of temperature on the free and
immobilized ␣-CGTase. The optimum temperature of free and
3
.1.5. Adsorption time
The effect of adsorption time on the conversion of the immobi-
◦
◦
immobilized enzyme were 38 C and 45 C, respectively. In com-
parison with the free CGTase, the immobilized CGTase had a higher
optimal temperature and a broader temperature range of high
activity.
lized enzyme has been studied with SBA-15 as the carrier (Table 1).
As shown in Fig. 3A, the adsorption of enzyme on carrier increased
initially only up to 3 h, and AA-2G concentration reached maximal
at 3 h. It is possible that, part of the enzyme molecules could inter-
act with the groups in the pore, causing the enzyme molecules to
be retained at the orifice of the carrier. However, the remaining
enzyme molecules were required to resist more blocking effect to
enter the internal pore. With the extension of adsorption time, con-
version rate decreased due to the reversibility of the adsorption and
enzyme loss.
3.2.3. Reusability
Reusability is of considerable importance for various applica-
tions of biocatalysts in a commercial point of view. The enzyme
reuse means that the stability of the final enzyme preparation
should be high enough to permit this reuse. Therefore, the enzyme
needs to be very stable or to become highly stabilized during the

Z. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 223–229
227
Fig. 3. Effects of (A) SBA-15 adsorption time and (B) immobilization time on the conversion rate of the immobilized enzyme: (ꢀ) free enzyme and (ꢁ) immobilized enzyme.
Fig. 4. Effect of temperature on immobilized and free ␣-CGTase activities: (ꢀ) free
Fig. 6. Reusability of immobilized enzyme.
enzyme and (ꢁ) immobilized enzyme.
immobilization process. The immobilized enzyme retained 65% of
◦
its initial activity after 4 cycles of reuse at 45 C for 24 h (Fig. 6). This
result showed that, the immobilized enzyme can be easily recov-
ered and used repeatedly although significant loss of its activity.
Prousoontorn and Pantatan reported that the immobilized CGTase
retained its activity up to 74.4% of the initial catalytic activity
after being used for 3 cycles [2]. The reusability of the immobi-
lized CGTase in this work is a little lower in comparison with that
reported by Prousoontorn and Pantatan [2], and this needs to be
improved with further studies.
3.2.4. Operational stability
One of the most important parameters to be considered in
enzyme immobilization was operational stability. Stability deter-
mines the possible application of enzyme in large scale processes.
We produced AA-2G continuously using the immobilized ␣-CGTase
in sealed plastic tube. The operation conditions were selected on
the basis of the foregoing experimental results. Fig. 7 shows that
the maximal concentration of AA-2G reached 9.6 g/L and 12 g/L at
◦
3
8 C in the presence of free ␣-CGTase and immobilized ␣-CGTase,
◦
Fig. 5. Effect of pH on immobilized and free ␣-CGTase activities: (ꢀ) free enzyme (at
respectively. At the optimal temperature 45 C for the immobi-
◦
◦
◦
3
8 C), (᭹) immobilized enzyme (at 38 C) and (ꢁ) immobilized enzyme (at 45 C).
lized ␣-CGTase, AA-2G concentration reached 21 g/L, which was
2
-fold of that in the presence of free ␣-CGTase. The conversion

2
28
Z. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 223–229
4. Conclusions
Immobilization of ␣-CGTase was conducted with SBA-15 as the
adsorbent, glutaraldehyde as the cross-linking agent, and sodium
alginate as the carrier. Optimal immobilization conditions were as
following: GA 0.01% (v/v), SBA-15 2 g/L, CaCl 3 g/L, adsorption time
2
3
h, and immobilization time 1 h. In comparison with the free ␣-
CGTase, the immobilized ␣-CGTase had a similar optimum pH and
a higher optimum temperature. The excellent properties of the
immobilized enzyme are great for the production of AA-2G from
ascorbic acid and ␤-cyclodextrin. At the optimal temperature, the
◦
conversion rate of the substrates with immobilized enzyme (45 C)
◦
was 2-fold of that with the free ␣-CGTase (38 C). The highest yield
of AA-2G reached 21 g/L at 5 d with the immobilized ␣-CGTase.
The immobilized ␣-CGTase retained up to 65% of its initial catalytic
activity after being used for 4 cycles.
Acknowledgements
Fig. 7. The operational stability of immobilized enzyme: (ꢀ) free enzyme and (ꢁ)
immobilized enzyme.
This project was financially supported by the grant from the
Key Program of National Natural Science Foundation of China (No.
2
0836003), the National Science Fund for Distinguished Young
rate of the substrates with immobilized enzyme is 2-fold of that
Scholars of China (No. 20625619), the Fundamental Research
Funds for the Central Universities (No. JUSRP30901), 973 Project
with the free ␣-CGTase. The molar yield and productivity of AA-2G
−
1
−1
, respectively. And
were 0.22 (AA-2G/AA, mol/mol), 0.22 g L
for the immobilized CGTase in [2], the molar yield and productiv-
h
(2007CB714306) and 973 Project (2010CB535014).
−
1
−1
ity of AA-2G were 0.0073 (AA-2G/AA, mol/mol) and 0.01 g L
h ,
respectively. The mole yield and productivity of immobilized ␣-
CGTase on the synthesis of AA-2G was much higher than those of
the immobilized ␣-CGTase from Paenibacillus sp. A11 [2].
References
[
[
[
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Biochem. 60 (1996) 645–649.
3.2.5. Storage stability
[4] T. Shibuya, Y. Miwa, M. Nakano, T. Yamauchi, H. Chaen, S. Sakai, M. Kurimoto,
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[
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