Báo cáo khoa học: Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with c-cyclodextrin pptx

Molecular imprinting of cyclodextrin glycosyltransferases
from Paenibacillus sp. A11 and Bacillus macerans with
c-cyclodextrin
Jarunee Kaulpiboon
1,2
, Piamsook Pongsawasdi
3
and Wolfgang Zimmermann
2
1 Department of Pre-Clinical Science (Biochemistry), Faculty of Medicine, Thammasat University, Pathumthanee, Thailand
2 Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Germany
3 Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
Cyclodextrin glycosyltransferase (EC 2.4.1.19;
CGTase) catalyzes four different reactions: cyclization,
disproportionation, coupling, and hydrolysis. Cyclo-
dextrins (CDs, cyclic oligosaccharides of glucose resi-
dues) are formed by the intramolecular circularization
reaction, whereas a linear malto-oligosaccharide is
Keywords
cross-linked imprinted proteins; cyclodextrin
glycosyltransferase; cyclodextrins; molecular
imprinting; product specificity
Correspondence
W. Zimmermann, Department of
Microbiology and Bioprocess Technology,
Institute of Biochemistry, University of
Leipzig, Leipzig 04103, Germany
Fax: +49 341 97 36798
Tel: +49 341 97 36781
E-mail: wolfgang.zimmermann@
uni-leipzig.de

Website: chemie.
uni-leipzig.de/agz
(Received 3 November 2006, revised 11
December, accepted 13 December 2006)
doi:10.1111/j.1742-4658.2007.05649.x
Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of
cyclodextrins from starch by an intramolecular transglycosylation reaction.
To manipulate the product specificity of the Paenibacillus sp. A11 and
Bacillus macerans cyclodextrin glycosyltransferases towards the preferential
formation of c-cyclodextrin (CD
8
), crosslinked imprinted proteins of both
cyclodextrin glycosyltransferases were prepared by applying enzyme
imprinting and immobilization methodologies. The crosslinked imprinted
cyclodextrin glycosyltransferases obtained by imprinting with CD
8
showed
pH and temperature optima similar to those of the native and immobilized
cyclodextrin glycosyltransferases. However, the pH and temperature
stability of the immobilized and crosslinked imprinted cyclodextrin glyco-
syltransferases were higher than those of the native cyclodextrin glycosyl-
transferases. When the catalytic activities of the native, immobilized and
crosslinked imprinted cyclodextrin glycosyltransferases were compared, the
efficiency of the crosslinked imprinted enzymes for CD
8
synthesis was
increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased.
Comparison of the product ratios by high-performance anion exchange
chromatography showed that the native cyclodextrin glycosyltransferases
from Paenibacillus sp. A11 and Bacillus macerans produced CD

6
:CD
7
:
CD
8
: ‡ CD
9
ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of
reaction at 40 °C with starch substrates. In contrast, the crosslinked
imprinted cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and
Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and
17 : 14 : 49 : 20, respectively. The size of the synthesis products formed by
the crosslinked imprinted cyclodextrin glycosyltransferases was shifted
towards CD
8
and ‡ CD
9
, and the overall cyclodextrin yield was increased
by 12% compared to the native enzymes. The crosslinked imprinted cyclo-
dextrin glycosyltransferases also showed higher stability in organic sol-
vents, retaining 85% of their initial activity after five cycles of synthesis
reactions.
Abbreviations
A11, Paenibacillus sp. A11; BM, Bacillus macerans; CD, cyclodextrin; CGTase, cyclodextrin glycosyltransferase; CLIP, crosslinked imprinted
proteins; HPAEC, high-performance anion exchange chromatography; TNBS, 2,4,6-trinitrobenzene sulfonic acid.
FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS 1001
transferred to an acceptor sugar molecule in the dis-
proportionation reaction. CGTase also catalyzes the
opening of a CD and transfer of the linear malto-

oligosaccharide to an acceptor sugar molecule in the
coupling reaction. Furthermore, CGTase can catalyze
the hydrolysis of glycosidic linkages in starch [1]. The
end-products, especially CD
6
,CD
7
and CD
8
(a-CD,
b-CD and c-CD), are extensively used in the food, cos-
metic and pharmaceutical industries, owing to their
ability to form inclusion complexes with appropriate
guest compounds [1–5]. However, a major disadvan-
tage of the synthesis of CD by CGTases is that all
native enzymes usually produce a mixture of CD
6
,
CD
7
,CD
8
and large-ring CD (‡ CD
9
), making proces-
ses to separate each type of CD unavoidable. These
are time-consuming, cost-intensive and potentially
unsafe for the consumer and the environment. To
resolve these problems, attempts to construct CGTases
with higher product specificity have been made, using

information on the three-dimensional structures of the
enzymes and genetic engineering techniques [6–8].
However, until now, no CGTase with a product specif-
icity for a single CD has been reported. Recently, a
technique of crosslinking imprinted proteins (CLIP)
has been described [9,10]. With a combination of
imprinting and enzyme immobilization methods, this
technique can be used for the production of recogni-
tion sites with predetermined selectivity in the enzyme.
In the first step, the enzyme is derivatized with itaconic
acid anhydride and then imprinted with ligands such
as substrate analogs or inhibitors in aqueous medium
[10]. Subsequently, the manipulated enzyme conforma-
tion is fixed by polymerizing it in a water-free organic
solvent. The ligand is removed in the final step, and
the CLIP enzyme can be used either in aqueous med-
ium or organic solvent. The CLIP enzymes show
altered substrate or product specificity and enhanced
stability in high concentrations of organic solvents
[11]. CLIP enzymes are also more enantioselective than
the native enzyme [12]. Furthermore, they are insoluble
and can be separated and recycled many times,
increasing their productivity. These beneficial proper-
ties are especially useful in the areas of synthetic
organic chemistry, biomedical applications, and envi-
ronmental catalysis.
In this study, the modification of the product specif-
icity and stability of two CGTases at the level of the
mature protein is described. The native enzymes from
Paenibacillus sp. A11 (A11) and Bacillus macerans

(BM) form CD
7
and CD
6
as their major products,
respectively [13,14]. The imprinting of the enzymes
with CD
8
, resulting in high levels of the desired prod-
uct being formed, is reported.
Results and Discussion
To provide enough attachment points for crosslinking
of the enzymes, the CGTases were derivatized with ita-
conic anhydride in an aqueous medium. Free amino
groups of lysine, hydroxyl groups of tyrosine or sul-
fhydryl groups of cysteine were covalently coupled
with itaconic anhydride [12]. By varying the protein ⁄
itaconic anhydride ratio, different degrees of derivati-
zation of the CGTases were obtained. The remaining
activity of the enzyme depended on the degree of deri-
vatization (Table 1). The optimum ratio (w ⁄ w) of both
CGTases to itaconic anhydride was 1 : 5. The resulting
degrees of derivatization of the A11 and BM CGTases
determined by the 2,4,6-trinitrobenzene sulfonic acid
(TNBS) assay were 62% and 65%, respectively. The
remaining activities of the derivatized A11 and BM
CGTases were 90% and 77%, respectively. With a
ratio of 1 : 3, the remaining activity of both enzymes
was not significantly different from a ratio of 1 : 5, but
the derivatization degree was significantly lower. With

the use of higher ratios, higher levels of derivatization
were possible, but resulted in a further decrease in
activity. In previous reports on the CLIP technique,
Kronenburg et al. [12] succeeded in manipulating the
enantioselectivity of epoxide hydrolase with a derivati-
zation degree of 70%, whereas Peißker et al . [10]
reported a 60% derivatization degree as optimum, con-
sidering the remaining activity of the resulting deriva-
tized protease.
The derivatized A11 and BM CGTases were
imprinted with CD
8
and crosslinked to obtain the cor-
responding CLIP CGTases. The derivatized nonim-
printed enzymes were also crosslinked to obtain
immobilized enzyme preparations for comparison.
The effect of pH on the activity of the different
CGTase preparations from A11 and BM was
Table 1. Degree of derivatization of the CGTases from A11 and
BM obtained with different protein ⁄ itaconic anhydride ratios and
remaining cyclization activity.
Ratio
a
(w ⁄ w)
Paenibacillus sp. A11 Bacillus macerans
Derivatization
degree (%)
Remaining
activity (%)
Derivatization

degree (%)
Remaining
activity (%)
1 : 0 0 100 0 100
1:3 54 97 52 80
1:5 62 90 65 77
1:7 71 80 73 75
1:9 80 76 76 74
1 : 11 90 72 79 72
1 : 13 94 66 86 67
a
Protein ⁄ itaconic anhydride.
Molecular imprinting of glycosyltransferases J. Kaulpiboon et al.
1002 FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS
determined in the pH range 5–11, as shown in
Fig. 1A,B. The optimum pH for the cyclization activ-
ity of the native, immobilized and CD
8
-imprinted
CLIP CGTases from A11 and BM was found to be
6.0. The pH activity profiles of the CGTases were sim-
ilar, showing 60% activity at pH 5.0, and decreasing
activity at higher pH values in the range of 7–11.
However, the CGTase from A11 showed a broader pH
optimum, extending from 6.0 to 8.0. When the immo-
bilized and CD
8
-imprinted CLIP CGTases were com-
pared with the native enzymes, a higher activity in the
pH range from 8 to 11 was observed. This effect was

more pronounced with the CGTase from BM. The
immobilized and the CLIP CGTase from A11 were
more stable than the native enzyme in the pH ranges
from 3 to 6 and 8 to 11, whereas the immobilized and
CLIP CGTases from BM showed higher stability in
the ranges from 3 to 7 and 9 to 11 (data not shown).
The activities of the native, immobilized and CD
8
-
imprinted CLIP CGTases from A11 and BM were also
determined at different temperatures in the range 30–
80 °C. The optimum temperature for the cyclization
activities of the different enzyme preparations were 40–
50 °C for the A11 CGTase (Fig. 2A) and 60 °C for the
BM CGTase (Fig. 2B). The similar temperature
optima of the different forms indicate that there was
no loss of enzyme activity through imprinting, immo-
bilizing and crosslinking of the native enzyme. The
temperature stability of the immobilized and CLIP
CGTases from A11 and BM at 60 °C and 70 °C was
considerably higher than that of the native enzymes
(Fig. 3A,B). This could be explained by a stabilizing
effect of the covalent crosslinking of the enzymes.
The immobilized and CD
8
-imprinted CLIP CGT-
ases from A11 showed 30% higher stability in
phosphate buffer containing up to 50% ethanol or
Relative activity (%)Relative activity (%)
pH

0
4 5 6 7 8 9 10 11 12
456789101112
20
40
60
80
100
A
B
0
20
40
60
80
100
Fig. 1. Effect of pH on the native (dotted line), immobilized (solid
line) and CD
8
-imprinted CLIP CGTase (dashed line) activity at 40 °C.
The CGTases were from A11 (A) and BM (B). The buffers used
were 0.2
M potassium phosphate (pH 5.0–7.0) (s), Tris ⁄ HCl
(pH 7.0–9.0) (x), and glycine ⁄ NaOH (pH 9.0–11.0) (D).
0
20
40
60
80
100

0
20 30 40 50 60 70 80 90
20 30 40 50 60 70 80 90
20
40
60
80
100
A
B
Temperature (°C)
Relative activity (%) Relative activity (%)
Fig. 2. Effect of temperature on native (dotted line), immobilized
(solid line) and CD
8
-imprinted CLIP CGTase (dashed line) activity at
pH 6.0. The CGTases were from A11 (A) and BM (B).
J. Kaulpiboon et al. Molecular imprinting of glycosyltransferases
FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS 1003
cyclohexane compared to the native enzyme, whereas
the immobilized and CD
8
-imprinted CLIP CGTases
from BM showed 15% higher stability. As ethanol and
other cosolvents have been shown to increase the yield
of CD produced by CGTases, the high stability of the
CD
8
-imprinted CLIP CGTases in the presence of eth-
anol could be used to further increase the product

yields of the CLIP enzymes [15]. The effect of polar
cosolvents has been explained by suppression of the
intermolecular transglycosylation reaction, which cau-
ses partial degradation of the CD products formed
[16,17]. With nonpolar solvents, CDs could form an
insoluble complex, resulting in their continuous
removal from the reaction by precipitation and a shift
of the equilibrium in favor of CD formation [18].
The reuse stability of the immobilized and CD
8
-
imprinted CLIP CGTases, which is an important
factor in the utilization of immobilized enzymes in
large-scale applications, was also determined [19,20].
More than 80% of the initial immobilized and CLIP
CGTase activities from A11 and BM were retained for
up to five cycles of synthesis reactions.
Comparison of the products obtained from the
native, immobilized and CD
8
-imprinted CLIP CGTas-
es revealed that the native CGTases from A11 and
BM produced CD
6
:CD
7
:CD
8
: ‡ CD
9

in ratios of
15 : 65 : 20 : 0 and 43 : 36 : 21 : 0, respectively, after
24 h of reaction at 40 °C. In contrast, the CLIP
CGTases from A11 and BM imprinted with CD
8
pro-
duced CD in ratios of 15 : 20 : 50 : 15 and
17 : 14 : 49 : 20, respectively (Table 2). The CLIP
CGTases showed an increase in product specificity
towards preferential formation of CD
8
. In addition to
a higher yield of CD
8
, the CD
8
-imprinted CLIP
CGTases also produced a higher overall yield of CD
compared with the native CGTases (Table 2). The
immobilized and CD
8
-imprinted CLIP CGTases also
produced larger amounts of large-ring CDs (‡ CD
9
)
after 24 h of reaction at 40 °C (Fig. 4A,B). As shown
in Fig. 4C,D, large-ring CDs were predominantly pro-
duced during the first 30 min of the reaction. After
24 h, the amount of large-ring CDs was reduced,
owing to their conversion to smaller CDs. However,

the conversion of large-ring CDs obtained with immo-
bilized and CD
8
-imprinted CLIP CGTases was slower
than with the native CGTases, indicating that the
immobilized and CD
8
-imprinted CLIP enzymes had
decreased hydrolysis and coupling activity.
When the cyclization activities of the CGTase prepa-
rations were compared, a 10-fold increase in the cata-
lytic efficiency (k
cat
⁄ K
m
) of the CLIP CGTases from
A11 and BM was observed, resulting from an increase
in the turnover rate (k
cat
) and the binding affinity (K
m
)
(Table 3). The K
m
values of the CLIP CGTases in the
coupling reaction indicated stronger binding of the
CD
8
substrate, whereas the turnover rates (k
cat

) were
0
20
40
60
80
100
A
0
20
40
60
80
100
B
20 30 40 50 60 70 80
20 30 40 50 60 70 80
Temperature (°C)
Remaining activity (%) Remaining activity (%)
Fig. 3. Effect of temperature on native (dotted line), immobilized
(solid line) and CD
8
-imprinted CLIP CGTase (dashed line) activity.
The CGTases were from A11 (A) and BM (B). The incubation was
performed at pH 6.0 for 30 min.
Table 2. Yields and product ratios of the native, immobilized and
CD
8
-imprinted CLIP CGTases from A11 and BM.
CGTase preparation

Yield
(%)
Product ratio (%)
CD
6
CD
7
CD
8
‡ CD
9
A11 CGTase
Native 42 15 65 20 0
Immobilized 56 18 39 24 19
CLIP imprinted with CD
8
54 15 20 50 15
BM CGTase
Native 44 43 36 21 0
Immobilized 57 28 27 22 23
CLIP imprinted with CD
8
56 17 14 49 20
Molecular imprinting of glycosyltransferases J. Kaulpiboon et al.
1004 FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS
slower than with the native CGTases, resulting in
higher yields of CD
8
after long reaction times. This
result should, however, be interpreted with caution, as

the catalytic efficiency of the enzymes in the coupling
reaction was determined using cellobiose, which is not
the natural acceptor in the starch transglycosylation
reaction.
The accumulation of large-ring CDs after 24 h of
reaction of the immobilized and CD
8
-imprinted CLIP
CGTases with starch can be explained by their chan-
ged hydrolytic activities. The decreased k
cat
and overall
catalytic efficiency of both CLIP enzymes in the hydro-
lysis reaction clearly indicated their lower CD hydroly-
sis activity.
In summary, the CD
8
-imprinted CLIP CGTases had
significantly higher catalytic efficiency for CD
8
cycliza-
tion and lower efficiency for CD hydrolysis, whereas
their efficiency in the CD
8
coupling reaction was slightly
increased when compared with the native enzymes.
These results correspond to the observed higher yield of
CD
8
and large-ring CDs obtained with the CLIP

CGTases. Whereas the immobilization of the CGTases
alone resulted in increased yields of large-ring CDs,
through reduction of their hydrolysis activity, the
observed shift in product ratios of the CD
8
-imprinted
CLIP CGTases suggests that the molecular imprinting
had a pronounced effect on the structure of the active
site of the enzymes. Imprinting of the CGTases with CD
of different sizes should have similar effects on their
preferential formation, which could, however, be expec-
ted to be limited by the increasing flexibility of the ring
structures of the larger CDs.
rotceteD esnopser
rotce
t
eD esnopser
Native A11 CGTase
Immobilized
A11 CGTase
CLIP A11 CGTase
CD
8
-imprinted
Native BM CGTase
Immobilized
BM CGTase
CLIP BM CGTase
CD
8

-imprinted
6
7
8
10
9
11
15 24
6
7
8
10
9
11
15
24
AB
CD

e
snopser

rotceteD

e
sno
p
s
e
r

ro
t
ce
t
eD
Native A11 CGTase
Retention time (min)
Immobilized
A11 CGTase
CLIP A11 CGTase
CD
8
-imprinted
Immobilized
BM CGTase
CLIP BM CGTase
CD
8
-imprinted
Retention time (min)
Native BM CGTase
15
6
6
7
7
8
8
10

10
9
9
11 11
15
24
24
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Fig. 4. HPAEC analysis of CD synthesized by different CGTase preparations from A11 (A, C) and BM (B, D) at 40 °C for 24 h (A, B) and
30 min (C, D). The numbers above the peaks indicate the degree of polymerization of the CD.
J. Kaulpiboon et al. Molecular imprinting of glycosyltransferases
FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS 1005
When the yields of CD
8
obtained after different
reaction times with the enzyme preparations were com-
pared, most of the CD
8
was found to be formed dur-
ing the first 30 min of incubation (Fig. 5). After 24 h,
the CD
8
-imprinted CLIP CGTases produced 32%
(A11) and 25% (BM) CD
8
. In comparison to the
native enzymes, the yield of CD
8
increased four-fold

with the CLIP CGTase from A11, and three-fold with
the CLIP CGTase from BM, after 24 h of reaction.
The amount of CD
8
produced by the CLIP CGTase
from BM slowly increased during the 24 h reaction
time, whereas no further increase occurred after 6 h of
reaction with the CLIP CGTase from A11. The differ-
ences in the time course of CD
8
formation detected
depended on the type of CGTase used, and are in
accordance with previously reported results. Terada
et al. [21] observed that the amount of CD
8
increased
when a CGTase from Bacillus sp. A2-5a was incubated
with starch for a long period of time, as a result of the
conversion of large-ring CDs to smaller CDs. In con-
trast, longer reaction times with CGTase from the bac-
terial isolate BT3 resulted in a 10% decrease in CD
8
[22].
The highest yield (26% conversion to CD
8
) with the
CLIP CGTase from A11 was found when synthetic lin-
ear amylose (molecular mass 280 kDa) was used as
substrate (Fig. 6). Lower yields of CD
8

were obtained
with starches from potato, pea, and rice. Corn starch
(73% amylopectin) and corn amylopectin gave the
lowest yields, owing to their branched structure. Low
yields were also obtained with dextrins, glucose oligo-
saccharides of short chain length (degree of polymer-
ization 23), indicating the preference of the CGTases
for long chains of unbranched glucose polymers.
In conclusion, the CGTases from Paenibacillus sp.
A11 and B. macerans could be imprinted with CD
8
,
which is not the major CD produced by the native
enzymes. CD
8
was produced by the CD
8
-imprinted
and crosslinked CLIP CGTases at much higher levels
than by the native enzymes. Moreover, the CLIP
CGTases showed higher stability and yielded larger
amounts of total CD in the synthesis reactions.
Experimental procedures
Materials and enzymes
CD
6
,CD
7
,CD
8

, potato starch (molecular mass 296 kDa),
corn starch (molecular mass 340 kDa), rice starch, corn
Table 3. Comparison of the cyclization, coupling and hydrolysis activities catalyzed by native, immobilized and CD
8
-imprinted CLIP CGTases
from A11 and BM.
CGTase preparation
CD
8
-cyclization activity CD
8
-coupling activity CD
6)24
-hydrolysis activity
K
m
,
starch
(mgÆmL
)1
)
k
cat
(s
)1
)
k
cat
⁄ K
m

(mgÆmL
)1
Æs
)1
)
K
m, CD8
(mM)
k
cat
(s
)1
)
k
cat
⁄ K
m
(M
)1
Æs
)1
)
K
m, CD6)24
(mgÆmL
)1
)
k
cat
(s

)1
)
k
cat
⁄ K
m
(mgÆmL
)1
Æs
)1
)
A11 CGTase
Native 0.83 ± 0.03 1.6 · 10
2
1.9 · 10
2
0.90 ± 0.20 1.2 · 10
2
1.3 · 10
5
1.25 ± 0.03 3.2 · 10
1
2.9 · 10
1
Immobilized 0.50 ± 0.02 2.5 · 10
2
5.0 · 10
2
0.55 ± 0.02 1.1 · 10
2

2.0 · 10
5
1.08 ± 0.01 8.0 · 10
0
6.4 · 10
0
CLIP imprinted
with CD
8
0.21 ± 0.01 4.9 · 10
2
2.3 · 10
3
0.26 ± 0.01 1.0 · 10
2
3.8 · 10
5
0.48 ± 0.04 5.5 · 10
0
1.1 · 10
1
BM CGTase
Native 0.55 ± 0.02 6.7 · 10
1
1.2 · 10
2
1.60 ± 0.20 4.2 · 10
2
2.6 · 10
5

1.30 ± 0.02 1.6 · 10
1
1.4 · 10
1
Immobilized 0.50 ± 0.01 9.0 · 10
1
1.8 · 10
2
0.63 ± 0.04 2.1 · 10
2
3.3 · 10
5
1.11 ± 0.05 6.3 · 10
0
4.8 · 10
0
CLIP imprinted
with CD
8
0.20 ± 0.01 2.1 · 10
2
1.1 · 10
3
0.25 ± 0.04 9.4 · 10
1
3.8 · 10
5
0.59 ± 0.02 5.3 · 10
0
9.0 · 10

0
A
B
Incubation time (h)
Yield of CD
8
(% of starch) Yield of CD
8
(% of starch)
0
0 5 10 15 20 25
0 5 10 15 20 25
10
20
30
40
0
10
20
30
40
Fig. 5. Time course of CD
8
formation by native (m), immobilized (j)
and CD
8
-imprinted CLIP CGTases (s) from A11 (A) and BM (B).
Molecular imprinting of glycosyltransferases J. Kaulpiboon et al.
1006 FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS
amylopectin, dextrin (degree of polymerization 23), cellobi-

ose, BSA, phenolphthalein, itaconic anhydride, TNBS,
2,2¢-azobis(2-methylpropionitrile), ethylene glycol dimetha-
crylate, water-free cyclohexane and n-propanol were pur-
chased from Sigma-Aldrich Chemie GmbH (Munich,
Germany). Pea starch (degree of polymerization 4000) was
kindly provided by Emsland-Sta
¨
rke GmbH (Emlichheim,
Germany). Synthetic amylose with an average molecular
mass of 280.9 kDa was prepared by the method of Kitam-
ura et al. [23]. Standards of large-ring CD (CD
9
to CD
24
)
were kindly provided by T Endo, Hoshi University, Tokyo,
Japan. Rhizopus sp. glucoamylase was obtained from Toy-
obo Co., Ltd (Osaka, Japan). BM CGTase was obtained
from Amano Enzyme Inc. (Aichi, Japan) and had a specific
activity of 1003 UÆmg
)1
of dextrinizing activity [13]. A11
CGTase was purified using starch adsorption and ion
exchange chromatography (DEAE-Toyopearl 650M col-
umn; Tosoh Corporation, Tokyo, Japan). The enzyme had
a specific activity of 5000 UÆmg
)1
, as determined by its
dextrinizing activity [13].
CGTase assays and protein determination

Cyclization activity was determined as CD-forming activity
by the phenolphthalein method [24]. CGTase (2.5 lg) was
added to 0.6 mL of 2.0% (w ⁄ v) soluble potato starch in
0.2 m potassium phosphate buffer (pH 6.0). The reaction
mixture was incubated for 30 min at 40 ° C. The reaction
was stopped by boiling for 10 min. An aliquot (0.5 mL)
was incubated with 2.0 mL of a solution containing 1.0 mL
of 4 mm phenolphthalein in ethanol, 4 mL of ethanol and
100 mL of 125 mm Na
2
CO
3
in distilled water. The absorp-
tion was measured at 550 nm, and the amount of CD
7
formed was calculated using a calibration curve. One unit
of activity was defined as the amount of enzyme that pro-
duced 1 lmol of CD
7
per min. The CD
8
-forming activity
was determined by HPAEC.
The coupling activity was determined by incubating CD
8
as donor with 50 mm cellobiose as glucosyl acceptor at
40 °C. Potassium phosphate buffer, 50 mm (pH 6.0), was
added to obtain a total volume of 0.5 mL. CD
8
and cellobi-

ose were preincubated for 5 min at 40 °C. The reaction was
started by adding enzyme (2.5 lg). After 10 min, the reac-
tion was stopped by boiling for 10 min. Subsequently, Rhiz-
opus sp. glucoamylase (0.385 U) was added to convert
linearized oligosaccharides to glucose at 40 °C for 30 min.
The released reducing sugars were determined with the di-
nitrosalicylic acid method [25]. One unit of activity was
defined as the amount of enzyme that produced 1 lmol glu-
coseÆmin
)1
.
The hydrolysis activity of the CGTase was determined by
incubating the enzyme with a CD mixture (CD
6
to CD
24
,
kindly provided by M N Mokhtar, Leipzig University) at
40 °C for 10 min. Subsequently, Rhizopus sp. glucoamylase
(0.385 U) was added at 40 °C and incubated for 30 min.
The amount of glucose formed was determined by HPAEC.
One unit of activity was defined as the amount of enzyme
that produced 1 lmol glucoseÆmin
)1
.
All kinetic experiments were carried out at 40 °Cin
potassium phosphate buffer (pH 6.0). Lineweaver–Burk
diagrams of the initial velocity against substrate concentra-
tion were plotted, and kinetic parameters were determined
using enzfitter software (Biosoft, Cambridge, UK). A

reaction time of 30 min was used in the Lineweaver–Burk
experiments. By varying the reaction time with fixed sub-
strate concentration, it was confirmed that the reaction
velocity was linear at this time point.
The protein concentrations were determined according to
Bradford [26], using BSA as standard.
Analysis of cyclodextrins
HPAEC with pulsed amperometric detection was performed
using a DX-600 system (Dionex Corp., Sunnyvale, CA,
USA) to analyze and quantify the CD products. A Carbo-
pac PA-100 analytical column (4 · 250 mm; Dionex Corp.)
was used. A sample (25 lL) was injected and eluted with a
linear gradient of sodium nitrate (0–10 min, increasing from
0% to 4%; 10–12 min, 4%; 12–32 min, increasing from 4%
to 8%; 32–48 min, increasing from 8% to 9%; 48–59 min,
A
0
5
10
15
20
25
30
0
5
10
15
20
25
30

h
c
r
a
ts
elb
u
los

ot
a
t
oP
hcrats a
e
P
h
crats

n
r
o
C
hc
r
ats

e
ci
R

e
s
ol
y
ma
c
i
t
e
htnyS
ni
t
ce
p
o
l
y
ma

er
up
n
ro
C
)3
2=PD
(

n
i

rtxeD
B
Substrates
Yield of CD
8
(% ) Yield of CD
8
(% )
Fig. 6. Yield of CD
8
synthesized by the native (black bars), immobi-
lized (gray bars) and CD
8
-imprinted CLIP CGTases (white bars) with
different substrates at pH 6.0 for 30 min. The CGTases were from
A11 (A) and BM (B).
J. Kaulpiboon et al. Molecular imprinting of glycosyltransferases
FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS 1007
increasing from 9% to 18%; 59–79 min, increasing from
18% to 28%) in 150 mm NaOH containing 2% acetonitrile
with a flow rate of 1 mLÆmin
)1
. The amounts of CD
6
to
CD
24
were quantified by comparison with standard curves
of authentic CD
6

to CD
24
samples.
Derivatization of the CGTases by acylation with
itaconic anhydride
Six milligrams of A11 and BM CGTase in 10 mL of 50 mm
potassium phosphate buffer (pH 6.0) was acylated by using
various amounts of itaconic anhydride. The solution mix-
tures with different ratios of itaconic anhydride per mg of
protein were stirred at 4 °C for 60 min. The pH was monit-
ored and maintained at 6.0 with 3 m NaOH. Nonreacted
itaconic anhydride and other low molecular mass com-
pounds were removed by gel filtration (HiTrap desalting
column; Amersham Biosciences, Uppsala, Sweden) with dis-
tilled water as the eluent. The fractions containing CGTase
activity were combined and lyophilized.
Determination of free amino groups
of the CGTases
The relative amounts of amino groups of the native and
covalently derivatized CGTases were determined according
to Habeeb [27] and Hall et al. [28] with TNBS. The extent
of derivatization was calculated according to Shetty & Kin-
sella [29]:
Derivatization degree ð%Þ¼½1 ÀðA
der
=A
nat
Þ Â 100
where A
der

and A
nat
are the absorbance values obtained
with derivatized and native protein solutions, respectively.
To a sample (0.3 mL) of native or derivatized protein
solution (0.5 mgÆ mL
)1
), 0.3 mL of NaHCO
3
(4%) and
0.3 mL of TNBS (0.1%) were added. The samples were
placed in a thermomixer at 37 °C (1000 r.p.m.). After
60 min, 0.47 mL of 1 m HCl was added, and the absorption
was measured at 335 nm against a blank treated as above
but containing 0.3 mL of deionized water instead of the
protein solution.
Imprinting of the derivatized CGTases
Dry derivatized enzyme (30 mg) and CD
8
(54 mg) were dis-
solved in 1 mL of 10 mm potassium phosphate buffer
(pH 5.5). The mixture was incubated at 25 °C for 30 min.
The CGTase–CD
8
complex was precipitated by adding
4 mL of n-propanol () 20 °C) and kept on ice for 10 min.
The precipitate was collected by centrifugation at 13 520 g
for 15 min at 4 °C on a Hettich 46R with angle rotor (Het-
tich GmbH & Co. KG, Tuttlingen, Germany). The pellet
was washed with 1 mL of n-propanol () 20 °C), freeze-

dried, and kept at ) 20 °C.
Crosslinking of imprinted derivatized
CGTases
Imprinted derivatized CGTases (10 mg) were suspended in
1 mL of dry cyclohexane by using an ultrasonication bath
for 15 min. Four milligrams of 2,2¢-azobis(2-methylpropio-
nitrile) and 200 lL of ethylene glycol dimethacrylate were
added to the suspension. The radical polymerization was
initiated by UV irradiation (k ¼ 312 nm) at 25 °C for 2 h.
The resulting polymer was kept in a refrigerator at 4 °C for
12 h. The white polymer was washed with 2 mL of cyclo-
hexane and with 50 mm potassium phosphate buffer
(pH 6.0) (3 · 10 mL) and lyophilized. The protein amounts
and enzyme activities were monitored during the different
steps.
Effect of pH and temperature on native,
immobilized and CD
8
-imprinted CLIP CGTase
activity
Each enzyme preparation (2.5 lg of protein) was incubated
with 2% (w ⁄ v) soluble starch at various pH values and
temperatures, and the cyclization activity of the enzymes
was assayed by the phenolphthalein method. Potassium
phosphate (0.2 m), Tris ⁄ HCl (0.2 m) and glycine ⁄ NaOH
(0.2 m) were used as buffers for pH 5.0–7.0, 7.0–9.0 and
9.0–11.0, respectively. For determining the effect of tem-
perature on the enzyme activity, the reactions were per-
formed between 30 °C and 80 °C.
Effect of pH on native, immobilized and

CD
8
-imprinted CLIP CGTase stability
Each enzyme preparation (2.5 lg of protein) was incubated
at 4 °C for 24 h in 10 mm acetate buffer (pH 3.0–5.0),
potassium phosphate buffer (pH 5.0–7.0), Tris ⁄ HCl buffer
(pH 7.0–9.0), and glycine ⁄ NaOH buffer (pH 9.0–11.0). The
remaining cyclization activity was assayed by the phenol-
phthalein method. The results were expressed as a percent-
age of the highest activity determined, which was defined as
100%.
Effect of temperature on native, immobilized
and CD
8
-imprinted CLIP CGTase stability
The thermostability of the enzyme preparations was investi-
gated over the range 30–80 °C. Each enzyme preparation
(2.5 lg of protein) in 10 mm potassium phosphate buffer
(pH 6.0) was incubated at temperatures between 30 °C and
80 °C for 30 min, and the residual cyclization activity was
assayed by the phenolphthalein method. The results were
expressed as a percentage of the highest activity determined,
which was defined as 100%.
Molecular imprinting of glycosyltransferases J. Kaulpiboon et al.
1008 FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS
Stability of native, immobilized and
CD
8
-imprinted CLIP CGTases in organic
solvents

The organic solvent tolerance of the native, immobilized
and CLIP CGTases in ethanol and cyclohexane was
determined by incubating the enzyme preparations
(0.25 mgÆmL
)1
)at30°C on a shaker with 10 mm
phosphate buffer (pH 6.0) containing 10–50% of the
solvents. After 1 h of incubation, the residual
cyclization activity was assayed by the phenolphthalein
method.
Reuse stability of immobilized and
CD
8
-imprinted CLIP CGTases
The immobilized and CLIP CGTases were recovered after
a synthesis reaction, and analyzed for their remaining cycli-
zation activity during five cycles of synthesis reactions.
After each cycle, the enzymes were filtered off and washed
thoroughly with 10 mm potassium phosphate buffer
(pH 6.0).
Synthesis of CDs with native, immobilized and
CD
8
-imprinted CLIP CGTases
The native, immobilized and CLIP CGTases (2 U of cycli-
zation activity) were incubated with 2.5 mL of 4% (w ⁄ v)
soluble potato starch in 0.2 m potassium phosphate buffer
(pH 6.0) at 40 °C for 24 h. The reaction was stopped by
boiling for 10 min. Glucoamylase (10 lL, 38.5 UÆmL
)1

)
was added for 3 h to convert the linear oligosaccharides to
glucose. Subsequently, the glucoamylase was inactivated by
boiling for 10 min, and the reaction mixtures were analyzed
by HPAEC.
Substrate specificity of native, immobilized and
CD
8
-imprinted CLIP CGTases
Soluble potato starch, pea starch, corn starch, rice starch,
synthetic amylose, corn amylopectin and dextrin substrates
(2% w ⁄ v) in 0.2 m potassium phosphate buffer (pH 6.0)
were incubated with each CGTase preparation (2.5 lgof
protein) at 40 °C for 30 min in a total reaction volume of
0.6 mL. Each reaction mixture was then analyzed by
HPAEC.
Acknowledgements
JK was supported by a research fellowship of the
Alexander von Humboldt Foundation, Germany. We
thank M. N. Mokhtar, Leipzig University, for his
advice on HPAEC analysis.
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