Optimized synthesis of specific sizes of maltodextrin glycosides by the coupling reactions of Bacillus macerans cyclomaltodextrin glucanyltransferase

Article abstract of DOI:10.1016/j.carres.2005.11.014

Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase, EC 2.4.1.19), in reaction with cyclomaltohexaose and methyl α-d- glucopyranoside, methyl β-d-glucopyranoside, phenyl α-d- glucopyranoside, and phenyl β-d-glucopyranoside gave four kinds of maltodextrin glycosides. The reactions were optimized by using different ratios of the individual d-glucopyranosides to cyclomaltohexaose, from 0.5 to 5.0, to obtain the maximum molar percent yields of products, which were from 68.3% to 78.6%, depending on the particular d-glucopyranoside, and also to obtain different maltodextrin chain lengths. The lower ratios of 0.5-1.0 gave a wide range of sizes from d.p. 2-17 and higher. As the molar ratio was increased from 1.0 to 3.0, the larger sizes, d.p. 9-17, decreased, and the small and intermediate sizes, d.p. 2-8, increased; as the molar ratios were increased further from 3.0 to 5.0, the large sizes completely disappeared, the intermediate sizes, d.p. 4-8, decreased, and the small sizes, d.p. 2 and 3 became predominant. A comparison is made with the synthesis of maltodextrins by the reaction of CGTase with different molar ratios of d-glucose to cyclomaltohexaose.

Full text of DOI:10.1016/j.carres.2005.11.014

Carbohydrate
RESEARCH
Carbohydrate Research 341 (2006) 210–217
Optimized synthesis of specific sizes of maltodextrin glycosides
by the coupling reactions of Bacillus macerans
cyclomaltodextrin glucanyltransferase
Seung-Heon Yoon and John F. Robyt*
Laboratory of Carbohydrate Chemistry and Enzymology, Department of Biochemistry, Biophysics, and Molecular Biology,
4252 Molecular Biology Building, Iowa State University, Ames, IA 50011, USA
Received 19 August 2005; accepted 4 November 2005
Available online 2 December 2005
Abstract—Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase, EC 2.4.1.19), in reaction with cyclomaltohexaose and
methyl a-D-glucopyranoside, methyl b-D-glucopyranoside, phenyl a-D-glucopyranoside, and phenyl b-D-glucopyranoside gave four
kinds of maltodextrin glycosides. The reactions were optimized by using different ratios of the individual D-glucopyranosides to
cyclomaltohexaose, from 0.5 to 5.0, to obtain the maximum molar percent yields of products, which were from 68.3% to 78.6%,
depending on the particular D-glucopyranoside, and also to obtain different maltodextrin chain lengths. The lower ratios of 0.5–
1.0 gave a wide range of sizes from d.p. 2–17 and higher. As the molar ratio was increased from 1.0 to 3.0, the larger sizes, d.p.
9–17, decreased, and the small and intermediate sizes, d.p. 2–8, increased; as the molar ratios were increased further from 3.0 to
5.0, the large sizes completely disappeared, the intermediate sizes, d.p. 4–8, decreased, and the small sizes, d.p. 2 and 3 became
predominant. A comparison is made with the synthesis of maltodextrins by the reaction of CGTase with different molar ratios
of D-glucose to cyclomaltohexaose.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Synthesis of maltodextrin glycosides; Cyclomaltodextrin glucanyltransferase; Cyclomaltohexaose; Methyl a-D-glucopyranoside; Methyl b-
D-glucopyranoside; Phenyl a-D-glucopyranoside; Phenyl b-D-glucopyranoside; Transglycosylation reactions; Acceptor reactions; Coupling reactions;
Reaction optimization
1. Introduction
formed in the coupling reaction to give a series of malto-
dextrins of different sizes.1,4–6 In some instances, a
fourth hydrolytic reaction can occur when water acts
as an acceptor. This hydrolytic reaction had to have
occurred when B. macerans CGTase reacted with CD6
to give a series of cyclomaltodextrins of d.p. 7–11 and
maltodextrins d.p. 5–12 D-glucose residues.7 In the
presence of good acceptors, such as, D-glucose or methyl
a-D-glucopyranoside, the reaction of water with B. mac-
erans CGTase is much less favorable and the hydrolysis
reaction is very minor, if it occurs at all.7 More recently,
a CGTase from B. circulans was shown to have a spec-
ificity for primarily forming CD7, with lesser amounts of
CD6 and CD8.8 Kinetic studies with B. circulans
CGTase have shown that the enzyme has a specificity
for catalyzing the coupling reaction 13.7-times faster
using CD7 than using CD6,9 in contrast with the
Bacillus macerans cyclomaltodextrin glucanyltransferase
(CGTase) [EC 2.4.1.19] catalyzes three kinds of reac-
tions: (1) formation of a-(1!4)-linked cyclomaltodext-
rins, containing primarily six D-glucopyranose units,
CD6, but also forming cyclomaltodextrins containing
seven and eight D-glucose units (CD7 and CD8) from
starch;1,2 (2) ÔcouplingÕ or ÔacceptorÕ reactions, primarily
between cyclomaltohexaose (CD6) and various carbohy-
drate acceptors, such as D-glucose, maltose, sucrose, and
so forth;3 and (3) disproportionation reactions that take
place between two maltodextrin molecules that are
*
Corresponding author. Tel.: +1 515 294 1964; fax: +1 515 294
0453; e-mail: jrobyt@iastate.edu
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.11.014

S.-H. Yoon, J. F. Robyt / Carbohydrate Research 341 (2006) 210–217
211
coupling reaction catalyzed by B. macerans CGTase,
which almost exclusively uses CD6 as the donor.7
B. macerans CGTase catalyzed coupling reactions have
been used: to specifically synthesize 14C-reducing-end
labeled maltodextrins;1,6,10 to synthesize methyl a- and
b-maltodextrin glycosides;11 to synthesize maltodextrin
chains attached to the non-reducing end of acarbose;12
to synthesize glycosylated salicyl alcohol to give salicin
analogues;13 and to synthesize higher cyclomaltodextrins
containing 9–25 D-glucopyranose units.7 B. circulans
CGTase has also been used to synthesize maltodextrins.14
Previously, we reported on the qualitative and quantita-
tive distribution of maltodextrins and cyclomaltodextrin
products formed in the coupling reaction of B. macerans
CGTase between different molar ratios of D-glucose and
cyclomaltohexaose.7 The kinds of products formed and
their quantitative amounts varied, depending on the
ratios and concentrations of the reactants. Specific glyco-
sides of maltodextrins of varying degrees of polymeriza-
tion (d.p.) are difficult and tedious to synthesize
chemically, but with specific enzymes, such as CGTase,
the synthesis is relatively facile.
In the present study, we report on the optimization of
the B. macerans CGTase synthesis of specific sizes of
maltodextrin glycosides, containing methyl a and b
and phenyl a and b aglycones by using different molar
ratios of 0.5–5.0 of the a and b anomers of methyl
and phenyl D-glucopyranosides to cyclomaltohexaose
(CD6). These reactions are compared with the syntheses
of maltodextrins obtained when D-glucose was the
acceptor.
substrate solution (1,490 lL), containing 18 mM CD6
and 18 mM methyl a-D-glucopyranoside in 25 mM
imidazolium–HCl buffer (pH 6.0) was preincubated at
37 ꢁC for 10 min. The enzyme reaction was started by
adding 10 lL of CGTase to the substrate solution.
Aliquots (150 lL) of the digest were taken every 5 min
for 25 min and the reaction was stopped by heating in
boiling water for 5 min. Glucoamylase (100 mU) was
added to the heat treated CGTase digest and incubated
at 37 ꢁC for 20 min, and then heated in boiling water
for 5 min to stop the reaction. The concentration of
the glucose was determined by the micro glucose
oxidase–peroxidase method.18 One CGTase unit was
defined as the number of l moles of CD6 transferred
to methyl a-D-glucopyranoside per min at the given
conditions.
2.3. CGTase reaction conditions for the synthesis of
maltodextrin glycosides
CGTase (200 mU) was added to 200 lL of the substrate
solutions, containing 25 mM imidazolium–HCl buffer
(pH 6.0), methyl a-D-glucopyranoside (or other glyco-
sides) and CD6 in various molar ratios from 0.5 to
5.0. Two major concentrations of CD6 were used for
the reactions with methyl a-D-glucopyranoside and D-
glucose, 100 mM for molar ratios of 0.5–3.0 and
50 mM for molar ratios of 4.0 and 5.0; for the other gly-
cosides, only 50 mM CD6 was used. The reactions were
carried out at 37 ꢁC and 30 lL aliquots were taken at 1,
3, 6, 9, 12, and 24 h. Equilibrium was reached in 12 h,
but the reactions were allowed to go 24 h for analysis.
The reactions were stopped by heating in a boiling water
bath for 5 min, diluted 5-fold with water and analyzed
by TLC. The synthesis of the maltodextrin glycosides
was demonstrated by treating the digests with beta-amy-
lase (10 U/mL) for 24 h at 37 ꢁC and then analyzed by
TLC.
2. Experimental
2.1. Materials
B. macerans CGTase was obtained by growing B. mac-
erans ATCC 8517 on wheat bran medium and purified
by a modification of the method of Kobayashi et al.,15
as previously described.16 Barley beta-amylase [EC
3.2.1.2] was purchased from Megazyme International
(Wicklow, Ireland). CD6 and CD7 were obtained from
Ensuiko Sugar Co. (Yokohama, Japan) and were pure
by TLC analysis. Methyl a-D-glucopyranoside was
purchased from Eastman Kodak Co. (Rochester, NY)
and recrystallized twice in distilled water. Methyl b-D-
glucopyranoside, phenyl a-D-glucopyranoside and phen-
yl b-D-glucopyranoside were obtained from Sigma
Chemical Co. (St. Louis, MO) and found to be pure
by TLC. Other chemicals were of reagent grade.
2.4. Separation and quantitative determination of the
CGTase coupling reaction products by TLC analysis
The CGTase coupling reaction products were separated
by TLC, using the solvents and conditions given in
Table 1. Each digest (1 lL) was spotted on a 20 · 20 cm
Whatman silica gel TLC plate to give an 18 cm path
length for 2 ascents of the solvents given in Table 1
for the different reactions. The carbohydrates were visu-
alized by dipping the plate into a methanol solution,
containing 0.3% (w/v) N-(1-naphthyl)ethylenediamine
and 5% (v/v) H2SO4, dried and heated at 120 ꢁC for
10 min. Maltodextrin standards and methyl a-D-gluco-
pyranoside and cyclomaltodextrins (CD6, CD7, and
CD8) were used as TLC standards to locate the products
on the TLC plate. The maltodextrins were prepared by
the reaction of B. licheniformis alpha-amylase (EC
2.2. Assay of the activity of CGTase
CGTase activity was measured by a modification of the
method of Thoma et al.17 as previously described.16 The

212
S.-H. Yoon, J. F. Robyt / Carbohydrate Research 341 (2006) 210–217
Table 1. TLC conditions for the separation of the products from the
methyl a, b and phenyl a, b D-glucopyranoside acceptor reactions with
CD6 catalyzed by B. macerans CGTase
of the enzyme reaction digests were quantitatively deter-
mined by scanning densitometry of the visualized carbo-
hydrates on the TLC plate.19,20
Reaction substrates Solventa
volume
Types
of TLC
No. of 18 cm
path length
ascents
The densitometric method gives a linear and structur-
ally independent response for D-glucose and each of the
maltodextrins and maltodextrin glycosides. It allows the
determination of the relative weight percent (or relative
mole percent) of the individual maltodextrin or saccha-
ride in a mixture by dividing the individual densities by
the sum of the densities, and permits a quantitative
determination without the use of an absolute stan-
dard.19,20 The use of the relative weight percent or mole
percent is important, in that it reduces the error in the
analyses by eliminating the necessity of putting exactly
equal amounts of carbohydrate on each TLC spot. This
is particularly important in making comparisons
between each individual reaction digest, as is done in
this study.
proportions plates
Me-a-Glc + CD6
Me-b-Glc + CD6
Ph-a-Glc + CD6
Ph-b-Glc + CD6
85:25:55:50 Whatman K5
85:20:50:50 Whatman K6
85:20:50:55 Whatman K5
85:25:55:50 Whatman K5
2
2
2
2
a Solvent = MeCN/EtOAc/1-PrOH/H2O.
3.2.1.1) [Thermyl, Novo Industries, Copenhagen] with
maize starch followed by reaction with isoamylase (EC
3.2.1.68) [Sigma Chemical Co., St. Louis, MO]. The
addition of methyl and phenyl groups to D-glucose
and maltodextrins increases the TLC mobilities of the
glycosides with respect to the non-glycosides as shown
in Figure 1. The identities of the maltodextrins and the
maltodextrin glycosides were determined by comparing
their mobilities with D-glucose and methyl a-D-gluco-
pyranoside, respectively. The components and products
3. Results and discussion
3.1. CGTase synthesis of methyl a-maltodextrin
glycosides
The products of the reactions of CGTase with different
molar ratios of methyl a-D-glucopyranoside to CD6
are presented in Table 2 and Figure 1. At relatively
low molar ratios, R = 0.5 and 1.0, there was a significant
amount of CD7 and CD8 formed and relatively equal
molar amounts of Me-a-G2 to Me-a-G12 and apprecia-
ble amount of higher Me a-maltodextrin glycoside, d.p.
13 and higher. As the molar ratio was increased the
higher maltodextrin glycosides decreased. The inter-
mediate-sized maltodextrin glycosides, d.p. 9–4, reached
a maximum at a ratio of 2.0; thereafter, they decreased
and Me-a-G2 and Me-a-G3 reached a maximum at a
ratio of 4.0. CD7 and CD8 also continued to decrease
after a ratio of 1.0 and completely disappeared at a ratio
of 3.0 (see Table 1). A ratio of 5.0 gave a decrease in all
of the maltodextrin glycosides.
The CGTase reactions with Me-a-Glc and CD6
should not have given the formation of any maltodext-
rins. This was confirmed by reacting the CGTase digests
with beta-amylase, which gave maltose, Me-a-Glc to
Me-a-G3; no D-glucose or maltotriose was produced,
both compounds that should have been formed if malto-
dextrins had been synthesized (see the even numbered
lanes in Fig. 1).
Figure 1. Thin layer chromatogram of CGTase reaction products and
their reaction with beta-amylase. Lane 1, maltodextrin standards. Lane
2, cyclomaltodextrins (CD6, CD7, and CD8) and Me-a-Glc standards.
Lane 3, reaction with 50 mM Me-a-Glc and 100 mM CD6 (R = 0.5).
Lane 4, reaction of the products of lane 3 with beta-amylase. Lane 5,
reaction with 100 mM Me-a-Glc and 100 mM CD6 (R = 1.0). Lane 6,
reaction of the products of lane 5 with beta-amylase. Lane 7, reaction
with 200 mM Me-a-Glc and 100 mM CD6 (R = 2.0). Lane 8, reaction
of the products of lane 7 with beta-amylase. Lane 9, reaction with
300 mM Me-a-Glc and 100 mM CD6 (R = 3.0). Lane 10, reaction of
the products of lane 9 with beta-amylase. Lane 11, reaction of 200 mM
Me-a-Glc and 50 mM CD6 (R = 4.0). Lane 12, reaction of the
products of lane 11 with beta-amylase. Lane 13, reaction of 250 mM
Me-a-Glc and 50 mM CD6 (R = 5.0). Lane 14, reaction of the
products of lane 13 with beta-amylase.
3.2. CGTase synthesis of methyl b-maltodextrin
glycosides
TLC analysis showed that a series of methyl b-malto-
dextrin glycosides were formed. The results of the
reaction of CGTase with different ratios of methyl

S.-H. Yoon, J. F. Robyt / Carbohydrate Research 341 (2006) 210–217
213
Table 2. Percent molar composition of the products from the CGTase reactions with methyl a-D-glucopyranoside and cyclomaltohexaose in various
molar ratios
CD6 (mM)a
Me-a-Glc (mM)
Molar ratio (R)b
100c
50
100
70
0.7
100
100
1.0
100
200
2.0
100
300
3.0
50c
200
50
250
5.0
0.5
4.0
CD6a
CD7a
CD8a
13.65
9.87
5.31
10.79
8.03
4.34
7.06
6.03
2.43
2.74
0.66
1.41
1.53
1.20
0.55
—
—
—
—
—
—
Mole % CDs
28.83
23.16
15.53
4.81
1.53
1.20
0.55
Me-a-Glc
Me-a-G2
Me-a-G3
Me-a-G4
Me-a-G5
Me-a-G6
Me-a-G7
Me-a-G8
Me-a-G9
Me-a-G10
Me-a-G11
Me-a-G12
Me-a-G13
Higher Me-a-Gn
9.75
7.74
4.10
4.10
5.01
6.32
4.32
2.82
2.04
2.60
2.26
1.60
0.41
18.12
10.85
9.55
7.54
6.45
6.83
6.11
3.81
3.65
2.63
2.40
2.15
1.43
0.38
13.07
14.55
12.84
9.61
7.40
7.21
6.10
3.69
3.49
2.47
2.28
2.02
1.33
0.33
11.17
23.45
16.50
12.04
9.02
8.15
6.39
4.27
2.80
2.18
2.14
1.62
1.29
0.43
4.90
35.17
21.17
12.85
8.83
6.95
4.81
3.07
1.85
0.96
0.99
0.75
0.53
0.12
0.42
37.48
23.15
14.19
8.51
5.86
3.88
2.19
1.21
0.67
0.67
0.49
0.34
0.07
0.10
45.50
22.75
12.82
8.26
4.86
2.61
1.28
0.64
0.27
0.26
0.13
0.06
0.01
0.01
Mole % of Me-a-Gn
61.42
65.99
69.92
71.74
63.3
61.32
53.95
a CD6, CD7, and CD8 represent cyclomaltohexaose, cyclomaltoheptaose, and cyclomaltooctaose, respectively; Gn = n number of D-glucopyranoside
units.
b Molar ratio R = [Me-a-Glc]/[CD6].
c 1.0 unit/mL was used for 100 mM CD6 digests and 0.5 unit/mL was used for 50 mM CD6 digests.
b-D-glucopyranoside to CD6 are given in Table 3. A
maximum yield of 68.2 mol % of the maltodextrin glyco-
sides was obtained when the molar ratio was 1.0. As the
molar ratio was increased to 2.0, there was a significant
Table 3. Percent molar composition of the products from the CGTase reaction with methyl b-D-glucopyranoside and cyclomaltohexaose in various
molar ratios
CD6 (mM)a
Me-b-Glc (mM)
Molar ratio (R)b
100c
50
100
100
1.0
100
200
2.0
100
300
3.0
50c
200
50
250
5.0
0.5
4.0
CD6a
CD7a
CD8a
9.64
13.43
1.89
3.45
6.17
0.84
0.59
1.81
0.14
0.15
0.62
0.04
0.10
0.22
—
0.04
0.08
—
Mole % CDs
24.97
10.46
2.54
1.81
0.32
0.12
Me-b-Glc
Me-b-G2
Me-b-G3
Me-b-G4
Me-b-G5
Me-b-G6
Me-b-G7
Me-b-G8
Me-b-G9
Me-b-G10
Me-b-G11
Me-b-G12
Me-b-G13
Higher Me-b-Gs
11.10
11.19
5.24
4.76
5.50
3.70
3.33
2.61
2.37
1.67
3.19
3.57
3.16
13.63
21.30
16.38
8.73
8.51
6.81
2.91
3.24
2.99
2.41
1.79
2.13
2.12
1.84
8.38
34.99
23.06
11.21
7.56
6.01
2.30
2.83
1.88
1.39
1.03
1.22
1.09
0.97
1.94
45.67
23.12
9.41
6.31
4.95
2.04
2.18
1.27
0.94
0.64
0.83
0.60
0.53
0.69
55.59
21.49
7.61
5.39
3.78
1.67
1.58
0.70
0.46
0.31
0.40
0.32
0.23
0.18
57.47
21.22
7.70
5.32
3.50
1.64
1.39
0.53
0.31
0.18
0.26
0.17
0.11
0.09
Mole % Me-b-Gns
63.93
68.24
62.47
53.52
44.09
42.41
a CD6, CD7, and CD8 represent cyclomaltohexaose, cyclomaltoheptaose, and cyclomaltooctaose, respectively; Gn = n number of D-glucopyranoside
units.
b Molar ratio R = [Me-b-Glc]/[CD6].
c 1.0 unit/mL was used for 100 mM CD6 digests and 0.5 unit/mL was used for 50 mM CD6 digests.

214
S.-H. Yoon, J. F. Robyt / Carbohydrate Research 341 (2006) 210–217
decrease in the maltodextrin glycosides, d.p. 4 and
higher, although d.p. 4 and 5 were still significant, with
mole percents of 7.6 and 6.0, respectively. Me-b-G2 and
Me-b-G3 reached the maximum mole percents of 23.1
and 11.2, respectively, when the molar ratios were 2.0.
Ph-a-G2 was formed, with 16.4% Ph-a-G3 and 10.9%
Ph-a-G4 and very low amounts of CDs.
3.4. CGTase synthesis of phenyl b-maltodextrin
glycosides
3.3. CGTase synthesis of phenyl a-maltodextrin
glycosides
The results of the reaction of CGTase with different
ratios of phenyl b-D-glucopyranoside to CD6 are given
in Table 5. As with the previous syntheses, the largest
yields of the higher d.p. phenyl b-maltodextrin glyco-
sides were obtained at the lower molar ratios of 0.5.
The largest amounts (78.6%) of the phenyl b-maltodex-
trin glycosides were obtained with a molar ratio of 3.0,
giving significant amount of Ph-b-G2 to Ph-b-G5, with
Ph-b-G2 having a maximum amount of 34.1%. Rela-
tively low amounts of CDs were formed with a molar
ratio of 3.0 and higher.
The results of the reaction of CGTase with different
ratios of phenyl a-glucopyranoside to CD6 are given in
Table 4. Higher d.p. maltodextrin glycosides of the phe-
nyl series were separated by TLC up to d.p. 17. A molar
ratio of 0.5 gave the largest amount of the higher d.p.
products, although there was also a significant amount
(18.1%) of CD7 formed. The largest overall yield
(71.4%) of the maltodextrin glycosides was obtained
when the molar ratio was 1.0 and there were significant
amounts of d.p. 2–7 and 9 that were formed. With a
molar ratio of 2.0, there were significant amounts of d.p.
2–5 that were formed. As the molar ratio was increased
further, these same phenyl a-maltodextrin glycosides
were formed, with increased amounts of d.p. 2–4. At a
molar ratio of 5.0, the maximum amount (29.7%) of
3.5. CGTase synthesis of maltodextrins
The results of the reactions of CGTase with different
molar ratios of D-glucose and CD6 to give the synthesis
of maltodextrins are given in Table 6. The highest num-
ber of different sized maltodextrins, as well as the highest
Table 4. Percent molar composition of the products from the CGTase reaction with phenyl a-D-glucopyranoside and cyclomaltohexaose in various
molar ratios
CD6 (mM)a
Ph-a-Glc (mM)
Molar ratio (R)b
50c
25
50
25
0.5
50
25
0.5
50
25
0.5
50
25
0.5
50
25
0.5
0.5
CD6a
CD7a
CD8a
7.21
18.06
2.23
2.29
8.83
0.66
1.55
3.65
0.17
0.73
1.89
0.05
0.62
0.44
0.03
0.20
0.21
0.01
Mole % CDs
27.50
11.78
5.37
2.67
1.09
0.42
Ph-a-Glc
Ph-a-G2
Ph-a-G3
Ph-a-G4
Ph-a-G5
Ph-a-G6
Ph-a-G7
Ph-a-G8
Ph-a-G9
Ph-a-G10
Ph-a-G11
Ph-a-G12
Ph-a-G13
Ph-a-G14
Ph-a-G15
Ph-a-G16
Ph-a-G17
Higher Ph-a-Gs
13.95
9.13
12.97
9.56
7.15
2.43
1.95
0.46
2.46
1.42
1.48
0.94
0.74
1.13
1.11
1.05
0.34
4.20
16.85
16.17
18.07
15.41
9.76
2.85
1.83
0.60
2.23
0.86
0.81
0.51
0.30
0.47
0.36
0.32
0.08
0.64
26.27
22.83
16.73
12.65
8.87
2.19
1.55
0.40
0.95
0.51
0.39
0.24
0.13
0.19
0.11
0.09
0.02
0.11
27.27
24.45
19.08
13.54
8.61
1.79
1.08
0.50
0.49
0.24
0.18
0.08
0.03
0.02
—
34.78
27.49
16.60
11.41
5.98
1.14
0.70
0.13
0.29
0.17
0.10
0.06
0.03
0.04
—
35.90
29.69
16.37
10.86
4.94
0.89
0.47
0.15
0.18
0.07
0.03
0.02
—
—
—
—
—
—
—
—
—
—
—
—
Total Mole % Ph-a-Gnsa
58.55
71.37
68.36
70.09
64.13
63.69
a CD6, CD7, and CD8 represent cyclomaltohexaose, cyclomaltoheptaose, and cyclomaltooctaose, respectively; Ph-a-Gns = phenyl a-maltodextrin
glycosides, containing n number of D-glucose units.
b Molar ratio = [Ph-a-Glc]/[CD6].
c 0.5 U/mL of CGTase was added for 50 mM CD6 digests.

S.-H. Yoon, J. F. Robyt / Carbohydrate Research 341 (2006) 210–217
215
Table 5. Percent molar composition of the products from the CGTase reaction with phenyl b-D-glucopyranoside and cyclomaltohexaose in various
molar ratios
CD6 (mM)a
Ph-b-Glc (mM)
Molar ratio (R)b
50c
25
50
50
1.0
50
100
2.0
50
150
3.0
50
200
4.0
50
250
5.0
0.5
CD6a
CD7a
CD8a
9.77
12.82
3.13
5.54
9.72
1.06
2.58
4.25
0.26
1.30
2.05
0.10
0.78
1.30
0.06
0.56
0.77
0.02
Mole % CDs
25.72
16.32
7.09
3.44
2.15
1.35
Ph-b-Glc
Phb-G2
Ph-b-G3
Ph-b-G4
Ph-b-G5
Ph-b-G6
Ph-b-G7
Ph-b-G8
Ph-b-G9
Ph-b-G10
Ph-b-G11
Ph-b-G12
Ph-b-G13
Ph-b-G14
Ph-b-G15
Higher Ph-b-Gs
9.59
10.53
10.58
10.15
8.82
2.33
1.97
1.17
2.25
1.44
1.39
0.99
1.52
1.56
1.15
8.84
8.25
15.58
18.14
15.95
11.19
2.79
1.81
1.64
1.65
1.10
0.90
0.61
0.79
0.74
0.62
1.93
15.35
24.63
18.89
15.91
9.66
2.36
1.65
1.04
0.77
0.67
0.48
0.31
0.36
0.31
0.19
0.31
17.94
28.19
20.48
15.99
9.10
1.87
1.07
0.68
0.42
0.30
0.23
0.10
0.08
0.07
0.01
0.03
20.66
32.85
19.43
14.39
7.21
1.37
0.75
0.45
0.26
0.18
0.12
0.06
0.04
0.03
0.02
0.03
24.28
34.10
18.98
13.20
5.88
1.03
0.54
0.33
0.16
0.09
0.04
0.02
—
—
—
0.01
Mole % Ph-b-Gns
64.69
75.43
77.56
78.62
77.19
74.37
a CD6, CD7, and CD8 represent cyclomaltohexaose, cyclomaltoheptaose, and cyclomaltooctaose, respectively; Ph-b-Gns = phenyl b-maltodextrin
glycosides, containing n number of glucose units.
b Molar ratio = [Ph-b-Glc]/[CD6].
c 0.5 U/mL of CGTase was added for 50 mM CD6 digests.
Table 6. Percent molar composition of the products from the CGTase reactions with D-glucose and cyclomaltohexaose in various molar ratios
CD6 (mM)a
D-Glc (mM)
Molar ratio (R)b
100
50
0.5
100
100
1.0
100
200
2.0
100
300
3.0
50
200
4.0
50
250
5.0
CD6a
5.63
7.97
6.55
4.64
6.10
4.92
1.76
2.03
1.37
5.16
0.89
1.04
0.63
2.56
0.24
0.36
0.10
0.70
0.03
0.05
0.01
0.90
CD7a
CD8a–CD13
Total mole % CDs
20.15
15.66
D-Glc
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12 & higher
8.44
13.13
7.50
10.60
10.22
6.28
6.10
4.69
2.63
1.69
0.33
8.25
16.97
14.89
8.07
10.96
9.44
5.79
5.62
3.38
2.24
1.39
0.30
5.29
32.49
18.94
10.40
8.98
7.46
4.89
4.23
2.40
1.64
0.68
0.20
2.54
41.77
19.12
11.93
7.74
5.87
3.56
2.56
1.46
1.00
0.49
0.18
1.75
60.18
18.36
10.40
5.02
3.37
1.59
0.16
0.08
0.04
0.02
64.00
17.33
8.74
4.80
3.12
1.74
0.12
0.05
0.02
—
—
—
—
0.07
Total mole % of MDs
71.42
67.38
62.35
55.67
39.12
35.91
a CD6, CD7, and CD8 = cyclomaltohexaose, cyclomaltoheptaose, and cyclomaltooctaose, respectively; G2, G3, G4 = maltose, maltotriose,
maltotetraose, and so forth, respectively; MDs = maltodextrins.
b Molar ratio, R = [D-Glc/CD6].
c 1.0 U/mL CGTase was used for 100 mM CD6 digests and 0.5 U/mL was used for 50 mM CD6 digests.
yield of 71.4% was obtained with a molar ratio of 0.5.
This is in contrast to the syntheses of the four maltodex-
trin glycosides, where the highest number of different
sized maltodextrins was also obtained at molar ratios

216
S.-H. Yoon, J. F. Robyt / Carbohydrate Research 341 (2006) 210–217
of 0.5, but the highest yields of the maltodextrin glyco-
sides were obtained at the higher molar ratios: methyl
a-maltodextrin glycosides at R = 2.0; methyl b-malto-
dextrin glycosides at R = 1.0; phenyl a-maltodextrin gly-
cosides at R = 3.0; and phenyl b-maltodextrin glycosides
at R = 4.0. As the molar ratio of D-glucose to CD6 was
increased, the amounts of the higher d.p. maltodextrins
were decreased, as they were for the maltodextrin glyco-
sides but the overall yields of the maltodextrins were
decreased. At a molar ratio of 3.0, the highest amounts
of d.p. 2–8 were obtained. As the molar ratios were
increased to 4.0 and 5.0, the amounts of the maltodext-
rins were further decreased.
3.6. Concluding discussion
The mechanism for the coupling reactions catalyzed by
CGTase (see Fig. 2) involves first the binding of CD6
at the active site with the opening of the ring by the
enzyme and the formation of a covalent, enzyme inter-
mediate, between maltohexaose and the enzyme.16 The
acceptors, glucopyranoside glycosides in the present
Figure 2. Reaction steps in the B. macerans CGTase coupling (acceptor) reactions. A. Opening of the CD6 ring and the formation of a covalent
maltohexaosyl enzyme complex that reacts with Me-a-Glc acceptor to give Me-a-G7. B. Reaction of two molecules of Me-a-G7 with CGTase to give
the disproportionation reaction at low molar ratios of Me-a-Glc to CD6. C. Reaction of methyl a-maltodecaoside (Me-a-G10) to give the CGTase
synthesis of CD7 at low molar ratios of Me-a-Glc to CD6. D. Reaction of CGTase covalent maltodextrin–enzyme complexes with Me-a-Glc at high
molar ratios of Me-a-Glc to CD6 to give small maltodextrin glycosides, Me-a-G3 and Me-a-G2.

S.-H. Yoon, J. F. Robyt / Carbohydrate Research 341 (2006) 210–217
217
study, then bind adjacent to the covalent, enzyme–
maltohexaosyl unit, so that the C-4-OH of the glucoside
makes a nucleophilic, SN2 attack onto C-1 of the malto-
hexanoyl unit, giving an a-(1!4) linkage and the forma-
tion of a maltoheptaose glycoside. This first product
then undergoes a CGTase catalyzed disproportionation
reaction between two maltoheptaose glycoside mole-
cules to eventually give a series of large and small mal-
todextrin glycosides.1,4,5 At low molar ratios, the large
maltodextrin glycosides are substrates for the formation
of the higher cyclomaltodextrins, CD7 and CD8. At
higher molar ratios, the large maltodextrin glycosides
decrease and hence the formation of the cyclomaltodext-
rins also decreases. It should be noted that for B. macer-
ans CGTase, CD6 is the preferred donor in the coupling
reactions, and CD7 and CD8 are very poor donors.7
The formation of the glycosides of the maltodextrins
gives a more stable material than maltodextrins in that
the most reactive group in the maltodextrins is the hemi-
acetal, reducing end that can undergo a variety of reac-
tions, such as oxidation, reduction, isomerization,
derivatization, and so forth. The formation of the glyco-
sides fixes the hemiacetal into an acetal with a specific a
or b configuration that cannot undergo anomerization
or other reactions. In different applications, the a or b
anomers could each have advantages. The a anomer is
more reactive than the b anomer in some kinds of reac-
tions. For example, the a anomer is more easily hydro-
lyzed, whereas the C-2 position of the b anomer is more
reactive than the C-2 position of the a anomer. Some
enzymes can hydrolyze the a anomer but not the b
anomer, and vice versa. Likewise, the methyl and phenyl
aglycone groups could each have their own advantages,
with the methyl group being a small alkyl group and the
phenyl group being a much larger aromatic ring. Pure
individual maltodextrin glycosides can be obtained by
charcoal–Celite column chromatography on a relatively
large, preparative scale.21 Columns as large as
15 cm · 1.5 m, have successfully been used.
4.0–5.0, an enhancement of the smaller sized maltodex-
trin and maltodextrin glycosides with d.p. values of 2
and 3 can be obtained. Further, molar ratios of 0.5–
1.0 gave significant, approximately equal mole percents
of maltodextrin glycosides of d.p. 2–10, for all four types
of the maltodextrin glycosides presented in this study,
along with significant amounts of the higher d.p. malto-
dextrin glycosides with d.p.s 12 and higher.
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