Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

Article abstract of DOI:10.1016/S0008-6215(98)00276-6

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an α-(1→6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached α-(1→6) to D-glucose, D-mannose, D-galactose, and methyl α-d-glucopyranoside. With d-fructopyranose and d-xylopyranose, PTS was linked α-(1→5) and α-(1→4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of α-(1→3) and/or α-(1→4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked α-(1→4) to the glucose residue. α,α-Trehalose gave two major products with PTS linked α-(1→6) and α-(1→4). Maltitol gave two major products with PTS linked α-(1→6) and α-(1→4) to the glucopyranose residue. Raffinose gave two major products with PTS linked α-(1→6) and α-(1→4) to the D-galactopyranose residue. Maltotriose gave two major products with PTS linked α-(1→6) and α-(1→4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked α-(1→5) as the major product and D-glucitol gave PTS linked α-(1→6) as the only product. The structures of the transfer products were determined using thin layer-chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and ¹³C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was D-glucitol. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(98)00276-6

Carbohydrate Research 313 (1998) 235–246
Transglycosylation reactions of Bacillus
stearothermophilus maltogenic amylase with acarbose and
various acceptors
a,1
a
a
b
Kwan Hwa Park , Myo Jeong Kim , Hee Seob Lee , Nam Soo Han ,
a
b,
Doman Kim , John F. Robyt *
a
Department of Food Science and Technology and Research Center for New Bio-Materials in Agriculture,
Seoul National Uni6ersity, Suwon 441-744, South Korea
Laboratory for Carbohydrate Chemistry and Enzymology, Department of Biochemistry and Biophysics,
Iowa State Uni6ersity, Ames, IA 50011, USA
b
Received 10 December 1997; accepted 16 October 1998
Abstract
It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose
to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an a-(1“6)
glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the
digest gave transfer products in which PTS was primarily attached a-(1“6) to
D-glucose, D-mannose, D-galactose,
and methyl a- -glucopyranoside. With -fructopyranose and -xylopyranose, PTS was linked a-(1“5) and
D
D
D
a-(1“4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose,
lactose, and gentiobiose. Lesser amounts of a-(1“3) and/or a-(1“4) transfer products were also observed for these
carbohydrate acceptors. The major transfer product to sucrose gave PTS linked a-(1“4) to the glucose residue.
a,a-Trehalose gave two major products with PTS linked a-(1“6) and a-(1“4). Maltitol gave two major products
with PTS linked a-(1“6) and a-(1“4) to the glucopyranose residue. Raffinose gave two major products with PTS
linked a-(1“6) and a-(1“4) to the
linked a-(1“6) and a-(1“4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked a-(1“5) as the
major product and -glucitol gave PTS linked a-(1“6) as the only product. The structures of the transfer products
were determined using thin-layer chromatography, high-performance ion chromatography, enzyme hydrolysis,
D-galactopyranose residue. Maltotriose gave two major products with PTS
D
13
methylation analysis and C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and
cellobiose, and the weakest acceptor was -glucitol. © 1998 Elsevier Science Ltd. All rights reserved.
D
Keywords: Maltogenic amylase; Transglycosylation; Acceptor reaction; Acarbose; Isoacarbose
1
. Introduction
transfer reactions between a segment of a
donor and various kinds of acceptors. Usually
the transfer takes place from a specific donor
to a relatively large number of structurally
different acceptors. The specificity of the
transfer is dependent on the specific enzyme
used, which usually determines the configura-
tion of the glycosidic bond that is formed. The
The enzymatic synthesis of a wide variety of
oligosaccharides has been attained in vitro by
*
Corresponding author. Tel.: +1-515-2941964; fax: +1-
5
15-2940453; e-mail: jrobyt@iastate.edu
1
Also corresponding author. Tel.: +82-331-2902028; fax:
+
82-331-2941336; e-mail: parkkh@plaza.snu.ac.kr
0008-6215/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(98)00276-6

236
K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
structure of the acceptor also often plays a
role in determining the position of transfer for
the formation of the glycosidic bond [1,2].
Enzymatic methods of glycosylation have
many advantages over chemical methods of
synthesizing oligosaccharides. For example,
complicated and tedious procedures for the
specific protection and deprotection of hy-
droxyl groups are not required and the en-
zymes often only transfer to one or two
hydroxyl groups with the transfer to one of
them greatly exceeding the other. This leads to
fewer reaction steps, simpler purification pro-
cedures, and higher yields.
drate acceptors are studied and their struc-
tures reported.
2
. Experimental
Materials.—Acarbose was obtained from
Bayer (Leverkusen, Germany) through Dr Y.
Konishi at Osaka University, Japan. Accep-
tors were obtained from various commercial
sources and were available in the laboratory.
Methods.—Purification of recombinant
BSMA enzyme. Escherichia coli transformant
carrying the BSMA gene [16] was cultured in
a 3 L jar fermentor containing Luria–Bertani
medium [17] at 37 °C for 11 h. The culture
broth was centrifuged (8,000×g, 4 °C, 7 min)
and the cells were resuspended in 300 mL of
50 mM Tris–HCl buffer (pH 7.5). The cells
were sonicated at 4 °C for 10 min (Sonicator:
VC-600, Sonics & Materials Inc., Danbury,
CT, USA, output 3.5, duty cycle 60, pulse
ON), centrifuged (10,000×g, 4 °C, 15 min),
and the supernatant was used as crude
BSMA. The crude BSMA was fractionated
between a saturated concentration of 20% (w/
v) and 50% (w/v) ammonium sulfate at 4 °C.
It was dialyzed for 12 h using a cellulose
dialysis membrane tube (Sigma Chemical Co.,
St. Louis, MO, USA, MW cut off 10,000 Da)
against 50 mM Tris–HCl buffer (pH 7.5). The
dialyzed enzyme solution was loaded onto a
Q-Sepharose column (anion exchange, 3×15
cm) that was equilibrated with 50 mM Tris–
HCl buffer (pH 7.5). The sample was washed
with the same buffer at a flow rate of 7
mL/min, and bound proteins were eluted with
a linear gradient of 0–0.5 M NaCl in 400 mL
of the buffer. Active fractions from the Q-
Sepharose chromatography were loaded onto
a Mono-Q HR5/5 column (anion exchange;
Waters, 1.0×10 cm), washed with the buffer
at a flow rate of 1 mL/min, and eluted with a
linear gradient of 0–0.5 M NaCl in 60 mL of
the buffer. Active fractions from the second
Mono-Q HR5/5 chromatography were con-
centrated and dialyzed against 20 mM Tris–
HCl buffer (pH 7.5).
Many different oligosaccharides have been
synthesized by enzymatic transfer reactions
involving glucansucrases [1–3], cyclodextrin
glucanosyltransferase [4,5], alpha-amylase
[
[
6,7], neopullulanase [8], and sialyl transferase
9], to mention only a few.
Acarbose, a pseudotetrasaccharide that has
a pseudo sugar ring at the nonreducing end
4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclo-
hexen-1-yl] linked to the nitrogen of 4-amino-
,6-dideoxy- -glucopyranose (4-amino-4-de-
oxy- -quinovopyranose), which is linked
[
4
D
D
a-(1“4) to maltose (see Fig. 10 for the struc-
ture), is widely recognized as a potent in-
hibitor of several carbohydrases such as
a-glucosidase [10], glucoamylase [11], alpha-
amylase [12], and cyclodextrin glucanosyl-
transferase (CGTase) [13], to mention a few.
Acarbose is bound in the active sites of alpha-
amylase and CGTase in such a manner that
the a-(1“4) glycosidic linkages are not
cleaved and transglycosylation cannot occur
[
12]. The maltogenic amylase from Bacillus
stearothermophilus (BSMA) [14] and Bacillus
sp. I-5 cyclodextrinase (CDase) [15] on the
other hand did bind acarbose to cleave the
first glycosidic linkage and give transglycosyl-
ation of a pseudotrisaccharide (PTS) [see Fig.
1
0 for the structure]. Cha et al. [16] cloned the
BSMA gene and showed that it hydrolyzed
cyclomaltoheptaose, pullulan, starch, and
acarbose and exhibited transglycosylation ac-
tivity, forming both a-(1“6) and a-(1“4)
glucosidic linkages [14].
In the present study, the transfer products
of BSMA acting on acarbose and 17 carbohy-
Assay of enzyme acti6ity. Assay of BSMA
activity was carried out according to the pro-

K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
237
cedure of reducing sugar determination by
the dinitrosalicylate (DNS) method [18]. The
enzyme digest was composed of 250 mL of
ized by dipping into 0.3% (w/v) N-(1-naph-
thyl)-ethylenediamine, 5% (v/v) H SO in
MeOH, followed by heating at 120 °C for 10
min. The carbohydrates were quantitatively
determined on the TLC plate by densitome-
try [20].
Analysis of acceptor reaction products by
HPIC. The reaction mixture was mixed with
an equal volume of acetonitrile, boiled for 5
min, centrifuged at 12,000×g for 5 min,
and filtered using a membrane filter kit (0.2
mm pore diameter, Gelman Sciences); 20 mL
of the sample was applied to a Dionex Car-
bopak PA1 column (0.4×25 cm, 10 mm par-
ticle diameter) and eluted with a 0 to 30%
2
4
1
% (w/v) substrate solution (cyclomalto-hep-
taose, soluble starch, pullulan or acarbose),
60–220 mL of 50 mM sodium citrate buffer
pH 6.0), and 30–90 mL of enzyme solution.
The reaction mixture was prewarmed at
5 °C for 5 min, then the diluted enzyme
1
(
5
solution was added, and incubated for 30
min. The reaction was stopped by adding 1.5
mL of DNS solution. Absorbances were
measured at 575 nm using a spectrophoto-
meter. One unit (U) of BSMA hydrolyzing
activity was defined as the amount of en-
zyme that forms reducing sugars to give an
absorbance at 575 nm of 1.0 when the reac-
tion was carried out at 55 °C for 30 min.
Hydrolyis and transglycosylation reactions
of acarbose. The hydrolysis rate of acarbose
was determined by mixing 3.9 mM acarbose
in 50 mM sodium citrate buffer (pH 6.0)
with BSMA. The reaction was carried out at
(
v/v) of 600 mM NaOAc gradient and 150
mM NaOH at a flow rate of 1.0 mL/min.
Methylation analysis. The methylation was
carried out according to Mukerjea et al. [19].
1
3
NMR analysis. The C nuclear magnetic
resonance spectrum was recorded with JNM
LA-400 FT-NMR spectrometer (Jeol, Japan)
and Heteronuclear Multiple Bond Connectiv-
ity (HMBC) mode.
5
2
5 °C and samples were taken every 8 h for
4 h. The reaction was stopped by boiling
for 5 min. For the acarbose transfer reac-
tion, 10% (w/v) acarbose in 50 mM sodium
citrate buffer (pH 6.0) and 20% (w/v) accep-
tor carbohydrate were mixed and preincu-
bated 1 h at 55 °C. Ten units of BSMA per
mg acarbose were added to the mixture and
the reaction was carried out for 24 h at
3
. Results
TLC and HPIC chromatograms were ob-
tained for the acceptor reactions of BSMA
with acarbose and various acceptors (Figs.
1
complimented each other in their ability to
separate the various compounds in the reac-
tions. The TLC mobility of compounds hav-
ing a-(1“4) and a-(1“3) linkages was
faster than compounds having a-(1“6) link-
ages while the opposite was observed for
HPIC.
–4). The two types of chromatography
5
5 °C. The products were analyzed by high
performance ion chromatography (HPIC)
and/or thin layer chromatography (TLC) af-
ter 24 h of reaction.
Preparation of isoacarbose. For the prepa-
ration of isoacarbose, the reaction condition
was the same as that of the transfer reac-
tions except that the acceptor carbohydrate
Analysis of the acceptor reactions with
monosaccharides.—Fig. 1 shows the TLC
analyses of the products in the acceptor reac-
was
D-glucose. When the reaction was termi-
nated, the excess amount of glucose in the
reaction mixture was removed by yeast fer-
mentation and the product was purified by
gel filtration chromatography on Bio-Gel P-2
column (2×90 cm).
tions with monosaccharides. When
D
-glucose
was added as an acceptor, one major and one
minor product were formed, 3 and 4, respec-
tively in lane B of Fig. 1. Methylation analysis
of the acceptor products (lane E in Fig. 9)
Analysis of acceptor reactions. The reaction
was analyzed by TLC on Whatman K6F sil-
ica gel plates (Fischer Scientific) with 1:3:1
EtOAc–2-propanol–water. After irrigating
twice, the TLC plate was dried and visual-
gave 2,3,6-, 2,4,6-, and 2,3,4-tri-O-methyl-
glucose. The 2,3,6-tri-O-methyl- -glucose was
formed from the first glucopyranose residue of
PTS. The 2,3,4-tri-O-methyl- -glucose was
formed from the linkage of PTS to -glucose
D-
D
D
D

238
K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
in the major acceptor product, indicating that
the linkage was a-(1“6).
confirmed by HMBC, and the structure was
found to be {[4,5,6-trihydroxy-3-(hydroxy-
methyl)-2-cyclohexen-1-yl]4-amino-4-deoxy}-
The formation of a-(1“6) linkages was
1
3
confirmed by C NMR analysis. The major
product was isolated by column chromatogra-
a -
pyranosyl-(1“6)-
this a-(1“6)-linked acceptor product of
glucose, isoacarbose. The 2,4,6-tri-O-methyl-
-glucose, formed in lesser amount, was from
the minor acceptor product, indicating that
-glucose. These
D
- glucopyranosyl - (1 “ 4) - O - a -
D
- gluco-
D
-glucose. We have called
1
3
phy, and its C NMR spectrum was obtained
Fig. 5). The chemical shifts for the anomeric
D
-
(
carbons of acarbose are 100.3 and 100.4 ppm,
corresponding to an a-(1“4) linkage. The
chemical shifts of the anomeric carbons of the
acceptor product was 98.6 and 98.7 ppm, cor-
responding to an a-(1“6) linkage. The pres-
ence of the a-(1“6) linkage was also
D
PTS was linked a-(1“3) to
D
results were in agreement with those obtained
by HPIC (Fig. 4). The HPIC chromatogram
(Fig. 4) gave a distinct separation of two
Fig. 1. TLC of the acceptor reactions of acarbose with various acceptors catalyzed by Bacillus stearothermophilus maltogenic
amylase. MD=maltodextrin standards (G1–G7); lane A, reaction with acarbose; lane B, glucose acceptor reaction; lane C,
mannose acceptor reaction; lane D, galactose acceptor reaction; lane E, fructose acceptor reaction; lane F, xylose acceptor
reaction; lane G, methyl a-
D-glucopyranoside; lane H, acarbose and isoacarbose standards. 1=pseudotrisaccharide (PTS);
2
=acarbose; 3=isoacarbose; 4=minor glucose acceptor product; 5=minor mannose acceptor product; 6=major mannose
acceptor product; 7 and 8=minor galactose acceptor products; 9=major galactose acceptor product; 10=minor fructose
acceptor product; 11=major fructose acceptor product; 12=minor xylose acceptor product; 13=major xylose acceptor product;
1
4 and 15=acceptor products of methyl a-D-glucopyranoside.

K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
239
Fig. 2. TLC of the acceptor reactions of di- and tri-saccharides with acarbose. MD=maltodextrin standards (G1–G7); lane A,
maltose acceptor reaction; lane B, cellobiose acceptor reaction; lane C, lactose acceptor reaction; lane D, gentiobiose acceptor
reaction; lane E, a,a-trehalose acceptor reaction; lane F, sucrose acceptor reaction; lane G, raffinose acceptor reaction; lane H,
maltotriose acceptor reaction; lane I, acarbose and isoacarbose standards. 1=pseudotrisaccharide (PTS); 2=acarbose; 3=
isoacarbose; 4=maltose acceptor product; 5=maltose acceptor product; 6=maltose acceptor product; 7 and 8=cellobiose
acceptor products; 9 and 10=lactose acceptor products; 11, 12 and 13=gentiobiose acceptor products; 14 and 15=a,a-trehalose
acceptor products; 16 and 17=sucrose acceptor products; 18 and 19=raffinose acceptor products; 20–22=maltotriose acceptor
products; 23=maltotetraose (G4). a=maltose; b=cellobiose; c=lactose; d=gentiobiose; e=a,a-trehalose; f=sucrose; g=
raffinose; h=maltotriose.
peaks that corresponded to the minor prod-
ucts linked a-(1“4), acarbose, and a-(1“3),
nigeroacarbose. The component with the a-
was from the methyl a- -glucopyranoside
D
residue of the acceptor product. Because in
the methylation analysis the formation of
(
1“3) linkage migrates faster than products
2,3,6-tri-O-methyl- -glucose can arise from
D
with a-(1“4) linkages in HPIC, peaks 4
both the glucose residue of PTS and from the
glucose residue from an a-(1“4)-linked
product, the a-(1“4) glucose acceptor
product can only be detected by measuring the
relative intensities of 2,3,6- and 2,4,6-tri-O-
and 5 in Fig. 4 corresponding to the a-(1“
3
) and a-(1“4)-linked acceptors, respec-
tively.
Reaction of acarbose in the absence of any
added acceptor (lane A of Fig. 1) gave -glu-
D
methyl- -glucose. If only an a-(1“3)-linked
D
cose, PTS (1 in lane A of Fig. 1), and isoacar-
bose (compare 3 in lane A with 3 in lanes B
and H of Fig. 1). The acceptor reaction of
product is formed, the intensities of the two
methylated glucoses would be the same, but if
an a-(1“4)-linked product is also formed, the
methyl a-
D
-glucopyranoside also gave two
intensity of 2,3,6 - tri - O - methyl -
D
- glucose
products, 14 and 15 in lane G of Fig. 1.
Methylation analysis of 14 (see lane C in Fig.
would be greater than the intensity of 2,4,6-
tri-O-methyl- -glucose. The results showed
that 2,3,6-tri-O-methyl- -glucose had 35%
higher intensity than 2,4,6-tri-O-methyl- -glu-
D
9
) gave 2,3,6- and 2,4,6-tri-O-methyl-
D-glu-
D
cose. The 2,3,6-tri-O-methyl- -glucose comes
D
D
from the first glucopyranose residue of PTS
and possibly also from a minor product linked
cose, indicating that an a-(1“4)-linked accep-
tor product was formed. Methylation analysis
of 15 (lane D in Fig. 9) gave 2,3,6- and
a-(1“4) and the 2,4,6-tri-O-methyl- -glucose
D

240
K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
D-glucopyranoside gave acceptor products
that were similar to those of methyl a- -glu-
D
copyranoside (data not shown).
The acceptor reactions of
C of Fig. 1) and -galactose (lane D of Fig. 1)
were similar to those of -glucose. The major
product with -mannose is 6 in lane C and
from its chromatographic mobility it is postu-
-man-
D-mannose (lane
D
D
D
lated to have PTS linked a-(1“6) to
D
nose and the minor product, 5 in lane C, is
likewise postulated to have PTS linked a-(1“
3
). The major product with -galactose is 9 in
D
lane D and it is postulated to have PTS linked
a-(1“6) to -galactose and the minor prod-
ucts, 7 and 8 in lane D, are postulated to have
PTS linked a-(1“3) and a-(1“4),
respectively.
The major acceptor product of -fructose
11 in lane E of Fig. 1) is postulated to have
PTS linked a-(1“5) to -fructopyranose and
the minor product, 10 in lane E, to have PTS
linked a-(1“4) to -fructopyranose. This is
based on the fact that -fructose exists pri-
D
D
Fig. 3. TLC of the acceptor reactions of sugar alcohols with
acarbose. MD=maltodextrin standards (G1–G7); lane A,
(
xylitol acceptor reaction; lane B,
D
-glucitol acceptor reaction;
D
lane C, maltitol acceptor reaction; lane D, acarbose and
isoacarbose standards. 1=pseudotrisaccharide (PTS); 2=
acarbose; 3=isoacarbose; 4=xylitol acceptor product; 5=
D
D
-glucitol acceptor product; 6 and 7=maltitol acceptor
D
products; a=maltitol.
marily in the pyranose ring form in solution
and that by analogy with the acceptor reac-
tions of dextransucrase, the major product is
2
,3,4-tri-O-methyl- -glucose, indicating the
D
formation of both a-(1“4)- and a-(1“6)-
linked products. As with compound 14, the
D
-glucose linked a-(1“5) to
leucrose) and the minor product is
linked a-(1“4) to -fructofuranose (isomal-
tulose) [1,21]. The major product of -xylose
D
-fructopyranose
(
D-glucose
2
,3,6-tri-O-methyl-
first glucopyranosyl residue of PTS and the
,3,4-tri-O-methyl- -glucose indicates that
-glucopy-
D
-glucose was from the
D
D
2
D
(
13 in lane F of Fig. 1) is postulated to have
PTS is linked a-(1“6) to methyl a-
D
PTS linked a-(1“4) to
pyranose ring is the major form of
D
-xylopyranose (the
ranoside. The acceptor reaction of methyl b-
D-xylose in
solution) and the minor product (12 in lane F
of Fig. 1) is postulated to have PTS linked
a-(1“3) to
-Glucose, PTS, and isoacarbose were
formed in all of the acceptor reactions due to
the hydrolysis of acarbose to -glucose and
PTS, of which the latter is transferred to
-glucose to give isoacarbose. The principal
D-xylopyranose.
D
D
D
linkage formed in the transfer reactions of
PTS to the various monosaccharides was a-
(
1“6) with minor transfers to form a-(1“3)-
and/or a-(1“4)-linkages. The exceptions be-
ing the transfer to -fructose in which the
major linkage was a-(1“5) and the minor
D
linkage was a-(1“4), and the transfer to
D
-
Fig. 4. High performance ion chromatogram of the acceptor
xylose in which the major linkage was a-(1“
reactions of glucose. 1=glucose; 2=pseudotrisaccharide
1
(
PTS); 3=isoacarbose; 4=3 -a-PTS-glucose; 5=acarbose.
4) and the minor linkage was a-(1“3).

K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
241
13
Fig. 5. C NMR spectrum of acarbose and the carbohydrate acceptor product produced by the action of BSMA on acarbose.
Analysis of the acceptor reactions with disac-
charides.—Fig. 2 shows the TLC analyses of
the compounds in the acceptor reactions with
various disaccharides. When maltose was the
acceptor (lane A of Fig. 2), the major acceptor
product was 6 with minor products 4 and 5
comes from both PTS and the reducing-end of
the cellobiose and is the major methylated
glucose produced.
The acceptor reaction with lactose gave one
major product (9 in lane C of Fig. 2) and a
minor product (10 in lane C of Fig. 2). The
major product has a TLC mobility less than
the major product of cellobiose, as would be
expected for a compound with PTS attached
(
Fig. 6); 3 was isocarbose formed in the trans-
fer of PTS to -glucose. When the maltose
acceptor digest was treated with pullulanase
D
(
(
lane F of Fig. 7), the major acceptor product,
6 in lane E of Fig. 7) completely disappears
a-(1“6) to the -galactose residue of lactose.
D
The minor product, 10, has not been identified
but could possibly be PTS linked a-(1“4) or
and PTS and maltose increase, indicating that
pullulanase is hydrolyzing an a-(1“6) linkage
a-(1“3) to the -galactose residue of lactose.
D
and the major acceptor product of maltose is
In the HPIC chromatographic analysis (data
not shown) of the lactose acceptor products,
the major product migrated faster than the
minor product, supporting the conclusion
above that the linkage of PTS to lactose is
a-(1“6).
2
6
-a-PTS-maltose (Fig. 8(A)).
The acceptor reaction with cellobiose indi-
cated a major acceptor product (7 in lane B of
Fig. 2) and a minor product (8 in lane B of
Fig. 2). Methylation analysis of the mixture of
acceptor products from the cellobiose reaction
The acceptor product of gentiobiose gave
one major product (13 in lane D of Fig. 2).
This acceptor product had a TLC mobility
that was considerably slower than that of the
a-(1“6)-linked acceptor product of lactose.
Gentiobiose is the b-(1“6) isomer of cel-
lobiose and would be expected to migrate
slower than either cellobiose or lactose and
hence its PTS acceptor product would also be
(
2
lane F of Fig. 9) gave 2,3,6-, 2,4,6-, and
,3,4-tri-O-methyl- -glucose with 2,3,4-tri-O-
methyl- -glucose in larger amounts than the
,4,6-tri-O-methyl- -glucose, indicating that
the major acceptor product 7 in lane B of Fig.
D
D
2
D
2
2
is 6 -a-PTS-cellobiose and the minor accep-
2
tor product 8 in lane B of Fig. 2 is 3 -a-PTS-
cellobiose. The 2,3,6-tri-O-methyl- -glucose
D

242
K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
Fig. 6. Reactions of BSMA with acarbose and maltose, forming the major acceptor products of pseudotrisaccharide (PTS) linked
a-(1“6) (major product) and a-(1“3) (minor product) to the nonreducing glucose residue of maltose. The formation of the
a-(1“4) linkage (very minor product) is not shown here.
expected to migrate slower than the PTS ac-
ceptor products of cellobiose and lactose. The
structure of the major gentiobiose acceptor
bility of these products and the specificity
pattern that has developed for the transfer
reactions of BSMA with acarbose, it is postu-
lated that the structure of the major raffinose
2
product is thus postulated to be 6 -a-PTS-gen-
Gal
tiobiose. The minor acceptor products of gen-
tiobiose, 11 and 12 in lane D of Fig. 2, are
postulated to have PTS linked a-(1“3) and
a-(1“4) to gentiobiose.
acceptor product is 6 -a-PTS-raffinose and
the structure of the minor acceptor product is
Gal
4
-a-PTS-raffinose.
The acceptor reaction with sucrose gave
two major products, 16 and 17 in lane F of
Fig. 2. The structures of the two sucrose ac-
ceptor products were determined using inver-
tase. Invertase is a b-fructofuranosidase that
specifically hydrolyzes the 2-b-fructofuranosyl
linkage of sucrose. Invertase reaction with the
sucrose acceptor products (4 and 5 in lane C
of Fig. 7) gave acarbose and isoacarbose (2
and 3 in lane D of Fig. 7), indicating that 4 in
lane C of Fig. 7 (16 in lane F of Fig. 2) had
PTS linked a-(1“4) to sucrose and 5 in lane
C of Fig. 7 (17 in lane F of Fig. 2) had PTS
linked a-(1“6) to sucrose (see Fig. 8(B,C) for
a summary of the reactions of invertase with
acceptor products of sucrose).
The acceptor reaction with a,a-trehalose
also gave two major products, 14 and 15 in
lane E of Fig. 2, similar to sucrose. From their
TLC mobility and similarity to the sucrose
acceptor products, the two a,a-trehalose ac-
ceptor products are postulated to have PTS
linked a-(1“4) and a-(1“6), respectively.
Analysis of the acceptor reactions with
trisaccharides.—The acceptor reactions with
raffinose gave one major product and one
minor product (19 and 18, respectively in lane
G of Fig. 2). From the chromatographic mo-
Fig. 7. TLC analysis of the reaction of the sucrose acceptor
product with invertase (lane D) and the reaction of the
maltose acceptor product with pullulanase (lane F). Lane A,
maltodextrin standards; lane B, 2=acarbose and 3=isoacar-
bose standards; lane C, acceptor products of sucrose, 1=
pseudotrisaccharide (PTS), 2=acarbose, 3=isoacarbose,
4
=sucrose acceptor product I, 5=sucrose acceptor product
II; lane D=sucrose acceptor products reacted with invertase,
1
6
=PTS, 2=acarbose, and 3=isoacarbose; lane E, 1=PTS;
=maltose acceptor reaction product; lane F, maltose accep-
tor product reacted with pullulanase, 1=PTS; lane G, 2=
acarbose and 3=isoacarbose standards.

K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
243
Fig. 8. Summary of the reactions of pullulanase and invertase with maltose and sucrose acceptor products. (A) Reaction of
pullulanase with the PTS-maltose acceptor product. (B) Reaction of invertase with the PTS-sucrose acceptor products, linked to
Glc
Glc
6
of sucrose and (C) reaction of invertase with the PTS-sucrose acceptor, linked to 3 of sucrose.
The acceptor reaction with maltotriose is
a-(1“3) or a-(1“4) and the major product,
7, has PTS linked a-(1“6) to the glucopyra-
nose residue.
complex in that maltotriose can be both an
acceptor for the transfer of PTS from acar-
bose and a hydrolytic substrate for BSMA
that gives glucose and maltose. The acceptor
reactions obtained with maltotriose had both
maltotriose acceptor products and maltose
and glucose acceptor products. This is seen in
lane H of Fig. 2 in which 20 is the major
maltose acceptor product (compare 20 in lane
H with 6 in lane A); 21 and 22 migrate more
slowly and are the acceptor products of mal-
totriose. The chromatographic mobilities of 21
and 22 indicate that PTS is linked a-(1“4)
and a-(1“6), respectively to maltotriose, in
about equal amounts.
Relati6e amounts of the acceptor products.—
The quantitative amounts of the acceptor
products were determined by quantitative
TLC densitometry [20]. Table 1 shows that
gentiobiose was the best acceptor. The relative
efficiencies of the individual acceptor reactions
were determined by summing the amounts of
products of each individual acceptor and di-
viding the sums by the sum of the products
from gentiobiose. The efficiencies of maltose
(99%) and cellobiose (92%) closely followed
that of gentiobiose.
D
-Glucose, methyl a-
D
-
glucopyranoside, -galactose, and
D
D
-xylose
Analysis of the acceptor reactions with sugar
alcohols.—Fig. 3 shows the TLC analysis of
the components in the acceptor reactions with
were relatively strong acceptors with efficien-
cies between 82 and 85%. Maltitol (69%),
D
-fructose (66%), lactose (66%), and
nose (66%) were moderate acceptors. Xylitol
(40%), a,a-trehalose (25%), and -glucitol
(16%) were relatively weak acceptors.
D
-man-
xylitol,
D-glucitol, and maltitol. Xylitol gave
one major product (4 in lane A of Fig. 3).
From the chromatographic mobility, it is pos-
tulated that PTS is linked a-(1“5) to xylitol.
D
The results indicate that many different car-
bohydrates were acceptors for the transfer of
PTS from acarbose by BSMA, and this en-
zyme favors an acceptor with a pyranose ring
structure. Further, the transfer is primarily to
a C-6, primary alcohol, to form an a-(1“6)
glucosidic linkage, but not exclusively so, sim-
ilar to the transfer acceptor reactions of dex-
D-Glucitol also gave one major product (5 in
lane B of Fig. 3) that is postulated to have
PTS linked a-(1“6) to -glucitol. The accep-
D
tor reaction with maltitol gave a major
product and a minor product (6 and 7, respec-
tively, in lane C of Fig. 3); it is postulated that
the minor product, 6, has PTS linked either

244
K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
transucrase in which the position of the trans-
fer reaction is partially dependent on the
structure of the acceptor [1].
4) linkages that could be hydrolyzed, the
products would accumulate. For example, if
the donor fragment from the transglycosyla-
tion was linked to the acceptor by an a-(1“3)
or a-(1“6) glycosidic linkage, the product
would not be hydrolyzed and would accumu-
late. In the present study, we analyzed the
acceptor products after reaction had occurred
for 24 h and equilibrium had been attained.
TVA transferred a glucosyl residue from the
donor (pullulan) to the acceptor molecules
with the formation of both a-(1“4) and a-
(1“6) linkages [7]. a-(1“3)-Linked acceptor
products were not observed. Similar observa-
tions have been reported for neopullulanase
[8] and B. licheniformis maltogenic amylase
[6]. In the present study of the acceptor reac-
tion of BSMA, the formation of a-(1“3) and
a-(1“6) linkages were observed and the prod-
ucts accumulated in significant amounts.
4
. Discussion
Tonozuka et al. [7] proposed that there
were two stages in the transglycosylation reac-
tion of Thermoactinomyces 6ulgaris R-47 al-
pha-amylase (TVA). The first is the
transglycosylation products followed by the
second, the hydrolysis of the accumulated
products. The hydrolysis of the transglycosyl-
ation products depends on the chemical struc-
ture of the acceptor molecules. If the acceptor
products contain a-(1“4)-linked glucose
residues, they would be readily hydrolyzed by
the alpha-amylase. However, if after hydroly-
sis, the resulting products do not have a-(1“
The reactions of BSMA with acarbose, with
acarbose and
and maltose, are summarized in Fig. 10. Acar-
bose is hydrolyzed to -glucose and PTS (re-
actions (A1) and (A2) in Fig. 10). When
-glucose is present in the reaction mixture,
PTS is transferred from acarbose to C-4, C-3,
and C-6 of -glucose to give acarbose,
D-glucose, and with acarbose
D
D
D
nigeroacarbose, and isoacarbose (reactions B1,
B2 and B3, respectively, in Fig. 10). When
other carbohydrates, such as maltose, are
present, PTS is transferred to C-6 or C-3 of
the nonreducing end glucose residue (reactions
(C1) and (C2), respectively, in Fig. 10). BSMA
can also transfer PTS to C-4 of the nonreduc-
ing glucose residue of maltose (reaction (C3)
in Fig. 10) and then hydrolyze the second
a-(1“4) glucosidic linkage of acarbose, giving
maltose and PTS (reaction (D) in Fig. 10).
When other acceptors are added, BSMA
cleaves the first a-(1“4) glucosidic linkage of
acarbose and transfers PTS to the acceptors,
primarily forming a-(1“6) linkages between
PTS and the acceptors, although a-(1“3) and
a-(1“4) linkages are also formed. With some
Fig. 9. TLC separation of the methylation analysis from
acarbose transfer products. Lane A, O-methylated glucoses as
standards; lane B, O-methylated sugars from acarbose; lane
C, O-methylated sugars from the acceptor reaction product of
methyl a-
O-methylated sugars from the acceptor reaction product of
methyl a- -glucopyranoside (15 in lane G of Fig. 1); lane E,
D-glucopyranoside, (14 in lane G, Fig. 1); lane D,
D
O-methylated sugars from the acceptor reaction mixture of
glucose in which the unreacted glucose and PTS were re-
moved using Bio-Gel P-2 column; lane F, O-methylated
sugars from the acceptor reaction mixture of cellobiose in
which PTS and the unreacted cellobiose and acarbose were
removed from the mixture, using Bio-Gel P-2 column chro-
matography.
acceptors, such as
D
-fructopyranose and -xy-
D
lopyranose, a-(1“5) and a-(1“4) linkages
are formed, respectively. The principal linkage
was to a primary alcohol, if it was present on
the acceptor.

K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
245
Table 1
Products of the transfer reaction of Bacillus stearothermophilus maltogenic amylase with acarbose and acceptors
a
b
c
d
Acceptor
PTS acceptor products (mg/mL)
Relative efficiency (%)
a-(1“6)
a-(1“5)
a-(1“4)
a-(1“3)
Total
D
-Glucose
7.40
9.27
4.20
5.43
5.55
8.84
8.20
1.82
1.81
(1.88)
9.21
11.15
7.39
9.22
7.37
10.35
11.24
1.82
4.51
2.76
4.60
7.45
6.02
6.32
9.55
7.77
9.39
82
99
66
82
66
92
100
16
40
25
41
66
54
56
85
69
84
Maltose
D
D
-Mannose
-Galactose
(3.19)
(1.45)
(1.82)
(2.34)
Lactose
Cellobiose
Gentiobiose
(1.5)
(1.80)
(1.24)
D
-Glucitol
Xylitol
a,a-Trehalose
Raffinose
(2.66)
(5.04)
(0.77)
(1.17)
(1.87)
(2.41)
4.05
(3.63)
6.54
(3.93)
2.90
(1.08)
(1.59)
2.73
D
-Fructose
Sucrose
Maltotriose
1.97
2.69
D
-Xylose
(3.01)
2.20
Maltitol
Methyl a-D-glucopyranoside
3.84
4.29
a
PTS is the pseudotrisaccharide produced by the cleavage of the first glucosidic linkage.
The structures of the acceptor products in parenthesis were estimated by their relative mobilities on TLC and HPIC and were
b
not definitively determined.
c
The amounts of the acceptor products were determined by TLC densitometry [20].
The relative transfer efficiencies were determined by comparing the total amounts of the acceptor products to those of
d
gentiobiose, the best observed acceptor.
The crystallographic structures of amy-
lolytic carbohydrases, such as alpha-amylase,
CGTase, and glucoamylase complexed with
acarbose have been reported [11–13]. None of
these enzymes hydrolyzed the acarbose or pro-
duced transglycosylation products. The bind-
ing of the acarbose with the active sites of
these enzymes are such that the potentially
susceptible a-(1“4) glycosidic linkages of
acarbose are not favorably positioned for the
catalytic groups to produce cleavage. Brzo-
zowski and Davies [12] have speculated that
alpha-amylase might hydrolyze and produce
transglycosylation of acarbose if acarbose was
bound at subsites −3 to +1, placing the
a-(1“4) glycosidic bonds in position for
cleavage. Until the present report, the enzy-
matic hydrolysis of the a-(1“4) glycosidic
bonds of acarbose or the transglycosylation of
an acarbose fragment to acceptors has not
been reported. The binding of acarbose with
the subsites of BSMA must be such that the
first a-(1“4) glycosidic linkage is favorably
placed in conjunction with the catalytic
groups to produce hydrolysis and transglyco-
sylation. The acarbose must be so specifically
bound to the active site of BSMA that only
the first a-(1“4) glycosidic linkage is cleaved.
The second a-(1“4) glycosidic linkage of
acarbose apparently is not favorably posi-
tioned to give its cleavage and the formation
of maltose and the pseudo-disaccharide,
acarviosine. There is the possibility that the
PTS transfer products obtained in this study
might be specific inhibitors for various other
carbohydrases.
Acknowledgements
This work was supported by the Korea
Science
and
Engineering
Foundation
(KOSEF) through the Joint Research Pro-
gram between the Research Center for New
Bio-Materials in Agriculture at Seoul Na-
tional University, Korea and the Laboratory
of Carbohydrate Chemistry and Enzymology
at Iowa State University, USA. The authors

246
K. Hwa Park et al. / Carbohydrate Research 313 (1998) 235–246
Fig. 10. Summary of the reactions of Bacillus stearothermophilus maltogenic amylase with acarbose to give (A) hydrolysis, (B)
transfer of PTS to -glucose, and (C) transfer of PTS to maltose.
D
[
10] D.D. Schmidt, W. Frommer, B. Junge, L.M ller, W.
Wingender, E. Truscheit, in W. Creutzfeldt (Ed.), First
International Symposium on Acarbose, Excerpta Medica,
Amsterdam, 1982, pp. 5–15.
would like to thank Ra Moon-Jin for the
NMR analysis.
[
[
[
11] A.E. Aleshin, L.M. Firsov, R.B. Honzatko, J. Biol.
Chem., 269 (1994) 15631–15639.
12] A.M. Brzozowski, M.J. Davies, Biochemistry, 36 (1997)
10837–10845.
13] B. Strokopytov, D. Penninga, H.J. Rozeboom, K.H.
Kalk, L. Diikhyizen, B.W. Dijstra, Biochemistry, 34
References
[
[
[
[
[
1] J.F. Robyt, Ad6. Carbohydr. Chem. Biochem., 51 (1995)
51–157.
2] D. Fu, M.E. Slodki, J.F. Robyt, Arch. Biochem. Bio-
phys., 276 (1990) 460–465.
(
1995) 2234–2240.
1
[
[
14] G.J. Kang, M.J. Kim, J.W. Kim, K.H. Park, J. Agric.
Food Chem., 45 (1997) 4168–4172.
15] T.J. Kim, J.H. Shin, J.H. Oh, M.J. Kim, S.B. Lee, S.
Ryu, K. Kwon, J.W. Kim, E.H. Choi, J.F. Robyt, K.H.
Park, Arch. Biochem. Biophys., 353 (1998) 221–227.
16] H.J. Cha, H.G. Yoon, Y.W. Kim, H.S. Lee, J.W. Kim,
K.S. Kweon, B.H. Oh, K.H. Park, Eur. J. Biochem., 253
3] D. Fu, J.F. Robyt, Arch. Biochem. Biophys., 283 (1990)
3
79–387.
4] E. Norberg, D. French, J. Am. Chem. Soc., 72 (1950)
202–1205.
[
1
5] J.F. Robyt, in R.B. Friedman (Ed.), Biotechnology of
Amylodextrin Oligosaccharides, ACS Symp. Ser. 458,
Am. Chem. Soc, Washington, DC, 1991, pp. 98–110.
6] I.C. Kim, J.H. Cha, J.R. Kim, S.Y. Jang, B.C. Seo, T.K.
Cheong, D.S. Lee, Y.D. Choi, K.H. Park, J. Biol. Chem.,
(
1998) 251–262.
[17] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular
Cloning: A Laboratory Manual, Vol. 3, 2nd ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY,
1989, p. A1.
[
267 (1992) 22108–22114.
[
[
7] T. Tonozuka, H. Sakai, T. Ohta, Y. Sakano, Carbohydr.
Res., 261 (1994) 157–162.
8] H. Takato, T. Kuriki, S. Okada, S. Takesada, M. Iizuka,
N. Minamimura, T. Imanaka, J. Biol. Chem., 267 (1992)
[18] C.L. Miller, Anal. Chem., 31 (1959) 426–428.
[19] R. Mukerjea, D. Kim, J.F. Robyt, Carbohydr. Res., 292
(1996) 11–20.
[20] J.F. Robyt, R. Mukerjea, Carbohydr. Res., 251 (1994)
187–202.
[21] J.F. Robyt, T.F. Walseth, Carbohydr. Res., 61 (1978)
433–445.
1
8447–18452.
9] M.D. Chappell, R.L. Halcomb, J. Am. Chem. Soc., 119
1997) 3393–3394.
[
(
.