Effect of the reaction temperature on the transglycosylation reactions catalyzed by the cyclodextrin glucanotransferase from Bacillus macerans for the synthesis of large-ring cyclodextrins

Article abstract of DOI:10.1016/j.tet.2003.10.112

The synthesis of cyclodextrins with from 6 to more than 50 glucose units by cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from Bacillus macerans was investigated. Analysis of the synthesized cyclic α-1,4-glucan products showed that a higher yield of large-ring cyclodextrins were obtained with a reaction temperature of 60°C compared to 40°C. The yield of large-ring cyclodextrins obtained at 60°C represented about 50% of the total glucans employed in the reaction. Analysis of the cyclodextrin-forming cyclization reaction and of the coupling reaction of the CGTase resulting in the degradation of mainly the larger cyclic α-1,4-glucans indicated higher rates of the cyclization reaction at 60°C compared to 40°C while the opposite was found for the coupling reaction.

Full text of DOI:10.1016/j.tet.2003.10.112

Tetrahedron 60 (2004) 799–806
Effect of the reaction temperature on the transglycosylation
reactions catalyzed by the cyclodextrin glucanotransferase from
Bacillus macerans for the synthesis of large-ring cyclodextrins
*
Qingsheng Qi, Xiaoyan She, Tomohiro Endo and Wolfgang Zimmermann
Department of Bioprocess Technology, Chemnitz University of Technology, D-09107 Chemnitz, Germany
Received 8 August 2003; revised 29 September 2003; accepted 17 October 2003
Abstract—The synthesis of cyclodextrins with from 6 to more than 50 glucose units by cyclodextrin glucanotransferase (CGTase, EC
2.4.1.19) from Bacillus macerans was investigated. Analysis of the synthesized cyclic a-1,4-glucan products showed that a higher yield of
large-ring cyclodextrins were obtained with a reaction temperature of 60 8C compared to 40 8C. The yield of large-ring cyclodextrins
obtained at 60 8C represented about 50% of the total glucans employed in the reaction. Analysis of the cyclodextrin-forming cyclization
reaction and of the coupling reaction of the CGTase resulting in the degradation of mainly the larger cyclic a-1,4-glucans indicated higher
rates of the cyclization reaction at 60 8C compared to 40 8C while the opposite was found for the coupling reaction.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
only been characterized recently (for a review, see Endo
et al. 2002).7 The molecular structure of CD9 resembles a
distorted boat shape because CD9 is more flexible than CD6,
Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is
an enzyme involved in the conversion of starch. The enzyme
is a member of the a-amylase family of glycosyl hydrolases
(family 13).1 CGTase can also hydrolyze glucan chains to
some extent similar to a-amylases, but differs in its ability to
form cyclodextrins (CD), cyclic a-1,4-glucans, as reaction
products. While CGTase has been previously employed for
the industrial production of CD6, CD7, and CD8 which are
composed of six, seven and eight glucose units (a-, b-, and
g-CD), the enzyme is also capable of synthesizing much
larger rings. The CGTases from various Bacillus species
including Bacillus macerans convert starch into a mixture of
CD with a degree of polymerization from 6 to more than
60.2,3
8,9
CD7 or CD8.
CD10 and CD14 have a butterfly-like
structure to reduce steric strain with twisting of some
glucose units to form flips and kinks.10–14 The structure of
CD26 contains two single helices with 13 glucose units each
in antiparallel direction.15–17 Due to their structural features
which are distinct from the small CD, large-ring CD could
find applications as novel host compounds in molecular
recognition processes.7
The formation of CD by CGTase proceeds by an a-retaining
double displacement mechanism.18,19 Two catalytic amino
acid residues, the catalytic acid/base residue Glu257 and the
nucleophile Asp229 are involved in the reaction (Fig. 1).
The glycosidic scissile bond oxygen of the bound a-1,4-
glucan donor is protonated by Glu 257 resulting in the
formation of an oxo-carbonium ion transition state. The
transition state collapses into a stable covalent glycosyl-
enzyme intermediate linked to Asp 229. The acceptor
activated by Glu257 acting as a base then attacks the
intermediate and a new a-glycosidic bond is formed via
another oxo-carbonium ion transition state (Fig. 1).
The synthesis and properties of industrially produced CD
have been studied intensively in the last years (for a review,
see Szejtli 1998).4 The molecular structures of CD resemble
a hollow, truncated cone with a hydrophobic cavity.5,6 Due
to this structure, CD can form inclusion complexes with
suitable guest molecules. Various properties of the guest
molecules, such as their water solubility, stability, and
bioavailability can thereby be manipulated. The commer-
cially available CD are therefore widely applied in the food
and pharmaceutical industries. The structure and properties
of large-ring CD composed of nine to 21 glucose units have
CGTase performs three transglycosylation reactions and a
hydrolysis reaction. Cyclic a-1,4-glucans are formed by an
intramolecular transglycosylation reaction where the term-
inal 4-OH group of the intermediate acts as an acceptor
(cyclization reaction, Fig. 2A). The reverse reaction also
occurs where the CD ring is opened and the linear
oligosaccharide is transferred to a linear glucan acceptor
(coupling reaction, Fig. 2B). Linear a-1,4-glucan products
Keywords: Cyclodextrin glucanotransferase; Large-ring cyclodextrins;
Transglycosylation reactions.
*
Corresponding author. Tel.: þ49-371-531-1581; fax: þ49-371-531-1790;
e-mail address: wolfgang.zimmermann@mb.tu-chemnitz.de
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.112

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Q. Qi et al. / Tetrahedron 60 (2004) 799–806
Figure 1. Scheme of the CGTase reaction mechanism. Reprinted from Uitdehaag, J.; van der Veen, B. A.; Dijkhuizen, L.; Dijkstra, B. W. Enzyme Microb.
Technol.. Catalytic mechanism and product specificity of cyclodextrin glycosyltransferase, a prototypical transglycosylase from the alpha-amylase family, 30
pp 295–304, Reprinted with permission. Copyright (2002) Elsevier.
In this study, we compare the effect of different reaction
temperatures on the cyclization and coupling reactions of
the CGTase from B. macerans influencing the yield of large-
ring CD synthesis products.
2. Results and discussion
Using synthetic amylose, a linear a-1,4-glucan with an
average molecular weight of 280.9 kDa as substrate, large-
ring CD are formed at an early stage of the reaction with
CGTases from Bacillus species.2,3,22 It has been observed
that the amount of large-ring CD decreased during a
prolonged incubation with the enzyme. The yield and size
Figure 2. Scheme of the transglycosylation reactions catalyzed by
CGTasep. Cyclization (A); coupling (B); disproportionation (C); and
hydrolysis (D). The dark circles represent glucose residues, the white
circles denote glucose residues with free reducing ends. pReprinted from
van der Veen, B. A.; van Alebeek, G. J.; Uitdehaag, J. C.; Dijkstra, B. W.;
Dijkhuizen, L. Eur. J. Biochem.. The three transglycosylation reactions
catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans
(strain 251) proceed via different kinetic mechanisms, 267 pp 658–665,
Reprinted with permission. Copyright (2000) Blackwell Publishing Ltd.
distribution of large-ring CD in the course of a synthesis
reaction will be strongly influenced by the extent of the
coupling and hydrolysis reactions by which the large-ring
CD are converted to smaller CD. Since CD7 and CD8 were
found to be poor substrates for the coupling and hydrolytic
reactions, they will accumulate during longer reaction
times.3
To further investigate the factors influencing the synthesis
of large-ring CD by CGTase, we compared the yield and
size distribution of CD obtained at different reaction
temperatures and incubation times. An analysis of the
cyclic a-1,4-glucan products obtained from synthetic
amylose as substrate catalyzed by the CGTase from B.
macerans is shown in Figure 3. Prior to analysis by high
performance anion exchange chromatography with pulsed
amperometric detection, the reaction mixtures were treated
with glucoamylase to convert non-cyclic glucans to glucose.
At a reaction temperature of 40 8C, large-ring CD were
detected after a reaction time of 1 h (Fig. 3A). After a
reaction time of 20 h, almost all of them were converted to
CD6, CD7 and CD8 as reported previously.2 In contrast, at a
reaction temperature of 60 8C, large-ring CD were formed
after an incubation time of 30 min and could still be
detected after a reaction time of 20 h (Fig. 3B). A time
course over 480 min of the total production of CD, of CD6–
CD8, large-ring CD, and of the reducing power at different
reaction temperatures is shown in Figure 4. The yield of
large-ring CD reached a maximum of 35% of the total
glucan after 2 h of incubation at 40 8C and decreased during
longer incubation times. Due to the conversion of the large-
ring CD to smaller CD by coupling or hydrolysis reactions,
their amount increased during longer incubation times
keeping the amount of total CD produced almost constant
(Fig. 4A). The maximum yield of large-ring CD after 2 h of
incubation at 60 8C reached about 50% of the total glucan
and remained almost constant during longer incubation
times (Fig. 4B).
are also formed in the third reaction, an inter-molecular
transglycosylation where a linear oligosaccharide is trans-
ferred to another linear glucan acceptor (disproportionation
reaction, Fig. 2C). In a hydrolysis reaction, a glucan is
cleaved and the reducing end is transferred to water
(Fig. 2D).
The amounts and size distribution of CD formed by CGTase
will be strongly influenced by the combined effects of the
three transglycosylation reactions, as well as by the
hydrolytic activity of the enzyme. CGTases isolated from
wild type bacterial strains have been shown to exhibit
different product profiles with respect to the size and amount
of the CD synthesized.20–22 A comparison of the large-ring
CD synthesis activity of CGTases from Bacillus spp. also
indicated that the enzymes exhibited different product
specificity. The CGTases differed in their hydrolytic
activities affecting the ratio of small CD to large-ring CD
products synthesized.3
The three transglycosylation reactions catalyzed by
CGTases show distinct differences in their kinetic mechan-
isms.3,23 The differences observed have been explained by
the way the substrates bind to the enzyme. Site-directed
mutagenesis and kinetic studies have suggested the
possibility to independently modify the cyclization and
coupling activities of CGTase.24 Thereby, the product range
of CGTase could be manipulated for the synthesis of CD of
specific sizes.

Q. Qi et al. / Tetrahedron 60 (2004) 799–806
801
Figure 3. HPAEC analysis of CD synthesized after 10, 30, 60, 120 and 1200 min by the CGTase from B. macerans at 40 8C (A) and 60 8C (B). Synthetic
amylose (0.5%) was incubated with 2 U ml21 CGTase. Peak numbers indicate the degree of polymerization of identified CD.
Beside the coupling reaction, the hydrolytic activity of
CGTase has also been considered to influence the conver-
sion of large-ring CD to smaller CD.3 However, it is difficult
to distinguish between the hydrolysis and the coupling
activity of the enzyme since it catalyzes both reactions at the
same catalytic site. After opening of the CD ring, the
resulting linear oligosaccharide will be transferred to water
in the case of hydrolysis. In the presence of other
oligosaccharides or glucose in the enzyme reaction mixture
these will also serve as acceptors resulting in a coupling
reaction. The amount of reducing power detected in the
reaction mixtures representing the amounts of reducing
sugars formed also depended on the reaction temperature. In
reaction mixtures incubated at 40 8C, it steadily increased
and reached higher levels compared to reactions performed
at 60 8C. The observed higher amounts of reducing sugars
formed at 40 8C may reflect the increased formation of
linear oligosaccharides due to hydrolysis reactions.

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Q. Qi et al. / Tetrahedron 60 (2004) 799–806
lower amounts of large-ring CD, and CD with a degree of
polymerisation above 13 could not be detected. A
comparison of the size distribution of all CD formed at
different temperatures also shows that the ratio of large-ring
CD to small CD was higher at 60 8C than at 40 8C (Fig. 5).
To further investigate the influence of the reaction
temperature on the amount and size of CD synthesized,
the optimum temperature for the cyclization and coupling
activities of the CGTase was compared. The results show an
optimum of the cyclization reaction at 60 8C, while the
optimum for the coupling activity with CD8 as substrate was
found around 45 8C (Fig. 6). A comparison of the kinetic
parameters of the cyclization reaction of the CGTase
showed a higher rate of CD7 formation at 60 8C compared
to 40 8C (Table 2). The Km value for synthetic amylose
could not be determined since the CD detection assay was
not sensitive enough to reliably measure the low amounts of
CD7 obtained at the low amylose concentrations required.
Analysis of the kinetic properties of the coupling activity of
the CGTases from B. circulans and Thermoanaerobacter-
ium thermosufurigenes has revealed a random-order reac-
tion mechanism involving a ternary complex, while the
CGTase from Bacillus sp. 1101 showed a non-sequential
ping-pong reaction mechanism.23,25–26 Our experimental
data for the CGTase from B. macerans also indicated a
random-order reaction mechanism. The Lineweaver–Burk
plots of the kinetic data obtained where the reciprocal of the
velocities was plotted against the reciprocal of the CD8
concentration at different acceptor concentrations did not
result in parallel lines (Fig. 7). In a random-order reaction,
any of the two substrates, CD8 and methyl-a-D-glucopyr-
anoside (MaDG), can bind first to the enzyme in a
sequential mechanism resulting in two apparent affinity
constants: Km in the absence and K0m in the presence of the
second substrate. The kinetic parameters determined at
different temperatures indicated higher rates of the coupling
reaction at 40 8C compared to 60 8C (Table 3). The affinity
constants K0m for both substrates in the presence of the other
substrate were lower at 40 8C than at 60 8C, while in the
absence of the second substrate the reverse effect was found.
The affinity for CD8 was decreased in the presence of the
acceptor at both temperatures. The decrease of affinity was
more pronounced at 60 8C compared to 40 8C. In contrast,
the affinity of the CGTase from B. circulans (strain 251) for
CD8 was not affected by the presence of the acceptor.23
Figure 4. Time course of the amounts of the total CD, CD6 to CD8, large-
ring CD and the reducing power produced by the CGTase from B. macerans
at 40 8C (A) and 60 8C (B). Total CD, B; CD6–CD8, X; large-ring CD, V;
reducing power, £. Synthetic amylose (0.5%) was incubated with 2 U ml21
CGTase.
The amounts of large-ring CD with a degree of polymeris-
ation from 9 to 21 synthesized during 4 h of incubation with
the CGTase at the two reaction temperatures were quantified
(Table 1). The amounts of each single large-ring CD formed
at 60 8C reached about 20–40 nmol ml21, corresponding to
conversion yields of 3–5% of the total glucan employed in
the reaction. Reactions performed at 40 8C yielded much
Table 1. Amounts of large-ring CD obtained by incubating 0.5% synthetic
amylose with CGTase from B. macerans at 40 and 60 8C
Structural studies of the CGTases from B. circulans strain
251 complexed with CD8 and of B. circulans strain 8
complexed with a CD7 derivative have indicated different
binding modes at the acceptor site for the CD and the linear
substrate.27 These differences may affect the binding of
linear oligosaccharides and CD at the active site of the
CGTase at different reaction temperatures favouring the
coupling reaction at the lower reaction temperature.
Cyclodextrin
40 8C (nmol ml21
)
60 8C (nmol ml21
)
CD9
34.66
11.43
4.63
4.70
2.11
nda
nd
nd
nd
nd
nd
45.71
49.03
44.33
45.35
41.59
29.74
18.88
33.58
45.33
33.48
39.68
24.26
30.25
CD10
CD11
CD12
CD13
CD14
CD15
CD16
CD17
CD18
CD19
CD20
CD21
The cyclization reaction of CGTase with amylose as
substrate has been previously described as an exo-type of
attack occurring at the sixth to the eighth glycosidic linkage
from the non-reducing end of a a-1,4-glucan chain.
However, recent results have shown that CGTases can
attack any a-1,4-linkage within the amylose molecule
nd
nd
a
nd, not detectable.

Figure 5. Size distribution of CD synthesized by the CGTase from B. macerans at 40 8C (open bars) and 60 8C (solid bars). Synthetic amylose (0.5%) was incubated with 2 U ml21 CGTase for 480 min.

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Q. Qi et al. / Tetrahedron 60 (2004) 799–806
Figure 6. Comparison of the coupling activity (V) and the cyclization activity (X) of the CGTase from B. macerans at different temperatures.
reaction was terminated by boiling the mixture for 10 min.
Glucoamylase (3.85 U ml21) was added and the mixture
was incubated for 24 h to convert the linear oligosacchar-
Table 2. Kinetic parameters of the CD8 cyclization reaction catalyzed by
the CGTase from B. macerans at 40 and 60 8C
ides to glucose. The amount of total CD in the sample was
calculated by subtracting the amount of glucose released by
glucoamylase from the total amount of glucose released by a
combined treatment with glucoamylase and a-amylase.3
Parameter
40 8C
60 8C
Vmax (U mg21
)
44.6^4.1
51.2^4.9
56.3^5.2
64.7^6.0
kcat (s21
)
resulting in the synthesis of a wide range of cyclic a-1,4-
glucans by a random cyclization reaction. By adjusting the
reaction conditions such as temperature and incubation
time, the yield of large-ring CD in the synthesis reaction can
be effectively increased.
3.3. Analysis of large-ring CD
High performance anion exchange chromatography with
pulsed amperometric detection (HPAEC-PAD) was carried
out using a DX-600 system (Dionex Corp., Sunnydale,
USA) to analyze and quantify the CGTase synthesis
products and to monitor the production process of large-
ring CD. A Carbopac PA-100 analytic column (4£250 mm,
Dionex Corp., Sunnydale, USA) was used. Samples (25 ml)
were centrifuged before analysis. Products were eluted with
a linear gradient of sodium nitrate (0–5 min, 1%; 5–49 min,
increasing from 1% to 18%; 49–89 min, increasing
from 18% to 35%; 89–100 min, increasing from 35% to
39%) in 200 mM NaOH containing 8% MeCN with a flow
rate of 1 ml min21. Retention times (min) of each CD
obtained were: CD6, 21.1; CD7, 27.3; CD8, 30.8; CD9, 36.3;
CD10, 34.7; CD11, 38,4; CD12, 44.3; CD13, 47.5; CD14, 49.8;
CD15, 51.6; CD16, 53.5; CD17, 53.0; CD18, 59.0; CD19, 61.2;
CD20, 62.9; CD21, 64.5). The amounts of CD6 to CD21
obtained were quantified by preparing standard curves
with concentrations of 0.1, 0.25, 0.5, 0.75 and 1 mM of each
CD.
3. Experimental
3.1. Chemicals and enzymes
Synthetic amylose with an average molecular weight of
280.9 kDa was prepared by the method of Kitamura et al.28
Soluble starch and a-amylase was from E. Merck AG
(Darmstadt, Germany) and methyl-a-D-glucose (MaDG)
was from Sigma-Aldrich Chemie GmbH (Munich,
Germany). Glucoamylase was from Toyobo Co., Ltd.
(Osaka, Japan) and B. macerans CGTase was from
Amano Enzyme Inc. (Aichi, Japan). The enzyme was
further purified using DEAE Sepharose Fast Flow media
(Amersham Biosciences Europe GmbH). The enzyme was
eluted with a linear gradient between 0 and 0.5 M NaCl in
Tris–HCl buffer, pH 7.8. Standards of large-ring CD (CD9
to CD21) were kindly provided by H. Ueda, Hoshi
University, Tokyo, Japan.
3.4. Assay for starch-hydrolyzing activity
The starch-hydrolyzing activity of the CGTase was
determined by the iodine method with some modifi-
cations.29 The reaction mixture (250 ml) contained 1.2%
soluble starch and 50 ml of the enzyme solution. After
incubation at 40 8C for 10 min, the reaction was stopped by
the addition of 500 ml of 0.5 M acetic acid–0.5 M HCl (5:1,
v/v). Then 5 ml of 0.005% I2 in 0.05% KI solution was
added to 100 ml of the mixture and the absorbance was
measured at 660 nm. One unit of the enzyme was defined as
the amount of enzyme producing a 10% reduction of the
intensity of the colour of the iodine complex per minute
under the conditions described.
3.2. Synthesis of large-ring CD
Synthetic amylose was dissolved in 10 ml of 90% DMSO
(v/v). To remove the DMSO, 0.5 ml of amylose solution
was mixed with 0.5 ml of distilled water and loaded on a
PD10 column (Amersham Biosciences Europe GmbH).
After eluting the column with 2 ml of distilled water, the
synthetic amylose was eluted with a further 1.5 ml of
distilled water and used immediately.3 The CD synthesis
reaction was performed using 0.5% synthetic amylose and
2 U ml21 CGTase in acetate buffer (50 mM, pH 5.5). The

Q. Qi et al. / Tetrahedron 60 (2004) 799–806
805
Figure 7. Lineweaver–Burk plots of the coupling reaction of the CGTase from B. macerans at 40 8C (A) and 60 8C (B). The reciprocal of the velocities is
plotted against 1/[CD8] at MaDG concentrations of 15 mM (X), 20 mM (W), 30 mM (P), and 60 mM (L).
Protein concentrations were determined using the Bradford
method30 and the reducing power by a modified Park–
Johnson method.31
ose (MW 280.9 kDa, 0.5%) as the substrate. The synthetic
amylose was incubated in 50 mM acetate buffer (pH 5.5)
with appropriately diluted CGTase. At time intervals (0.5–
1 min), 100 ml samples were taken and added to a mixture
containing 100 ml phenolphthalein in 800 ml sodium
hydroxide (0.03 M). One unit of activity was defined as
the amount of enzyme able to form 1 mmol of CD7 per min.
3.5. Cyclization activity
Cyclization activity was determined using synthetic amyl-

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Q. Qi et al. / Tetrahedron 60 (2004) 799–806
6. Lindner, K.; Saenger, W. Carbohydr. Res. 1982, 99, 103–105.
7. Endo, T.; Zheng, M.; Zimmermann, W. Aust. J. Chem. 2002,
55, 39–48.
Table 3. Kinetics parameters of the coupling reaction of CD8 with MaDG
catalyzed by the CGTase from B. macerans at 40 and 60 8C
Parameter
40 8C
60 8C
8. Fujiwara, T.; Tanaka, N.; Kobayashi, S. Chem. Lett. 1990,
739–742.
Km CD8 (mM)
Km0 CD8 (mM)
Km MaDG (mM)
Km0 MaDG (mM)
0.125^0.01
0.22^0.01
69.87^1.67
122.8^16.7
168.1^5.0
196.1^5.8
0.074^0.02
0.88^0.04
27.6^0.7
326.6^46.0
134.3^2.7
156.6^3.1
9. Harata, K.; Akasaka, H.; Endo, T.; Nagase, H.; Ueda, H.
Chem. Commun. 2002, 17, 1968–1969.
10. Ueda, H.; Endo, T.; Nagase, H.; Kobayashi, S.; Nagai, T.
J. Inclusion. Phenom. Mol. Recognit. Chem. 1996, 25, 17–20.
11. Harata, K.; Endo, T.; Ueda, H.; Nagai, T. Supramol. Chem.
1998, 9, 143–150.
Vmax (U mg21
)
kcat (s21
)
Kinetic analysis was performed with Sigmaplot software
(SPSS Inc., Chicago, USA) using the Michaelis–Menten
equation.
12. Jacob, J.; Geßler, K.; Hoffman, D.; Sanbe, H.; Koizumi, K.;
Smith, S. M.; Takaha, T.; Saegner, W. Angew. Chem. Rev.
1998, 37, 605–609.
13. Jacob, J.; Geßler, K.; Hoffman, D.; Sanbe, H.; Koizumi, K.;
Smith, S. M.; Takaha, T.; Saegner, W. Carbohydr. Res. 1999,
322, 228–246.
3.6. Coupling activity
The coupling activity between CD8 and methyl-a-D-
glucopyranoside (MaDG) was measured as described
previously.23 CD8 at concentrations of 1.5, 2, 2.5, 4 and
8 mM was used as donor substrate and up to 60 mM methyl-
a-D-glucopyranoside (MaDG) as acceptor substrate. The
substrates were incubated in 10 mM phosphate buffer (pH
5.5) for 5 min with appropriately diluted CGTase. The
reaction products were incubated with 3.85 U ml21 gluco-
amylase to convert the linear oligosaccharides formed to
glucose. The amount of glucose was determined with the
glucose oxidase method.32 One unit of enzyme activity was
defined as the amount of enzyme coupling 1 mmol of CD8 to
MaDG per min.
14. Endo, T.; Nagase, H.; Ueda, H.; Kobayashi, S.; Shiro, M. Anal.
Sci. 1999, 15, 613–614.
15. Gessler, K.; Uson, I.; Takaha, T.; Krauss, N.; Smith, S. M.;
Okada, S.; Sheldrick, G. M.; Saenger, W. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 4246–4251.
16. Nimz, O.; Gessler, K.; Uson, I.; Saenger, W. Carbohydr. Res.
2001, 336, 141–153.
17. Nimz, O.; Gessler, K.; Uson, I.; Laettig, S.; Welfle, H.;
Sheldrick, G. M.; Saenger, W. Carbohydr. Res. 2003, 338,
977–986.
18. Withers, S. G. Carbohydr. Polym. 2001, 44, 325–337.
19. Uitdehaag, J.; van der Veen, B. A.; Dijkhuizen, L.; Dijkstra,
B. W. Enzyme Microb. Technol. 2002, 30, 295–304.
20. Kobayashi, S. In Enzymes for Carbohydrate Engineering;
Park, K. H., Robyt, J. F., Choi, Y. D., Eds.; Elsevier:
Amsterdam, 1996; pp 23–41.
Kinetic analysis was performed with Sigmaplot software
(SPSS Inc., Chicago, USA). The following equation for a
random-order reaction mechanism was used to fit the
experimental data.33
21. Larsen, K. L.; Duedahl-Olesen, L.; Jørgens, H.; Christensen,
S.; Mathiesen, F.; Pedersen, L. H.; Zimmermann, W.
Carbohydr. Res. 1998, 310, 211–219.
v ¼ Vmax·a·b=ðK0mA·KmB þ KmB·a þ KmA·b þ a·bÞ
ð1Þ
v¼reaction rate, Vmax¼maximum rate; A and B¼donor and
acceptor substrate; a and b¼substrate concentrations; Km
and Km0 ¼affinity constants for the substrates in the absence
and in the presence and of the second substrate.
22. Zheng, M.; Endo, T.; Zimmerman, W. J. Inclusion Phenom.
2002, 44, 387–390.
23. van der Veen, B. A.; van Alebeek, G. J.; Uitdehaag, J. C.;
Dijkstra, B. W.; Dijkhuizen, L. Eur. J. Biochem. 2000, 267,
658–665.
24. Leemhuis, H.; Uitdehaag, J. C.; Rozeboom, H. J.; Dijkstra,
B. W.; Dijkhuizen, L. J. Biol. Chem. 2002, 277, 1113–1119.
25. Leemhuis, H.; Dijkstra, B. W.; Dijkhuizen, L. Eur. J. Biochem.
2003, 270, 155–162.
Acknowledgements
We thank Amano Enzyme Inc., Aichi, Japan for a gift of the
CGTase from B. macerans and H. Ueda, Hoshi University,
Tokyo, Japan for a gift of large-ring CD standards.
26. Nakamura, A.; Haga, K.; Yamane, K. FEBS Lett. 1994, 337,
66–70.
27. Schmidt, A. K.; Cottaz, S.; Driguez, H.; Schulz, G. E.
Biochemistry 1998, 37, 5909–5915.
References and notes
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T. Denpun Kagaku 1989, 36, 257.
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