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

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-

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

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

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
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

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,

) 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

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

-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

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

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

(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

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

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-

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-

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

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

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

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

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

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