Two novel oligosaccharides formed by 1F-fructosyltransferase purified from roots of asparagus (Asparagus officinalis L.)

Article abstract of DOI:10.1271/bbb.66.1419

Two novel oligosaccharides, tetra-and penta-saccharides were synthesized by fructosyl transfer from 1-kestose to 4^G-β-D- galactopyranosylsucrose with a purified 1^F-fructosyltransferase of asparagus roots and identified as 1^F-β-D-fructofuranosyl-4 ^G-β-D-galactopyranosylsucrose, O-β-D-fructofuranosyl-(2 → 1)-β-D-fructofuranosyl-O-[β-D-galactopyranosyl-(1 → 4)]-α-D-glucopyranoside and 1^F(1-β-D-fructofuranosyl) ₂-4^G-β-D-galactopyranosylsucrose, [O-β-D-fructofuranosyl-(2 → 1)]₂-β-D-fructofuranosyl- O-[β-D-galactopyranosyl-(1 → 4)]-α-D-glucopyranoside, respectively. Both oligosaccharides were scarcely hydrolyzed by carbohydrase from rat small intestine. Human intestinal bacterial growth by 1 ^F-/β-D-fructofuranosyl-4^G-β-D- galactopyranosylsucrose was compared with that by the tetrasaccharides, stachyose and nystose. Bifidobacteria utilized 1^F-β-D- fructofuranosyl-4^G-β-D-galactopyranosylsucrose to the same extent as stachyose or nystose. On the other hand, the unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcus faecalis, that produce mutagenic substances did not use the synthetic oligosaccharide.

Full text of DOI:10.1271/bbb.66.1419

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Two Novel Oligosaccharides Formed by 1F-
Fructosyltransferase Purified from Roots of
Asparagus (Asparagus officinalis L.)
Akira YAMAMORIa, Shuichi ONODERAa, Masanori KIKUCHIa & Norio SHIOMIa
a Department of Food Production and Utility Development, Graduate School of Dairy
Science Research, Rakuno Gakuen University Ebetsu 069-8501, Japan
Published online: 22 May 2014.
To cite this article: Akira YAMAMORI, Shuichi ONODERA, Masanori KIKUCHI & Norio SHIOMI (2002) Two Novel
Oligosaccharides Formed by 1F-Fructosyltransferase Purified from Roots of Asparagus (Asparagus officinalis L.), Bioscience,
Biotechnology, and Biochemistry, 66:6, 1419-1422, DOI: 10.1271/bbb.66.1419
To link to this article: http://dx.doi.org/10.1271/bbb.66.1419
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Biosci. Biotechnol. Biochem., 66 (6), 1419–1422, 2002
Note
Two Novel Oligosaccharides Formed by 1F-Fructosyltransferase Puriˆed
from Roots of Asparagus (Asparagus o‹cinalis L.)
†
Akira YAMAMORI, Shuichi ONODERA, Masanori KIKUCHI, and Norio SHIOMI
Department of Food Production and Utility Development, Graduate School of Dairy Science Research,
Rakuno Gakuen University, Ebetsu 069-8501, Japan
Received January 15, 2002; Accepted January 30, 2002
Two novel oligosaccharides, tetra-and penta-saccha-
rides were synthesized by fructosyl transfer from 1-ke-
terium (B.) longum, B. adolescentis and other strains
of Biˆdobacteria.1) We have also studied the puriˆca-
tion and characterization of several fructosyltran-
stose to 4G-
b
-
D
-galactopyranosylsucrose with a puriˆed
1F-fructosyltransferase of asparagus roots and identiˆed
as 1F- -fructofuranosyl-4G-
-galactopyranosylsu-
crose, -fructofuranosyl-(2
syl- -[ -galactopyranosyl-(1
side
topyranosylsucrose, [
sferases,
sucrose:sucrose
1-fructosyltransferase
b
O
-
D
b
-
D
(SST), 1F-fructosyltransferase (1F-FT) and the new
enzyme, 6G-fructosyltransferase (6G-FT),6) from
asparagus roots. We have previously found that
asparagus 1F-FT catalyzed fructosyl transfer from 1-
kestose to the non-reducing fructosyl residue ter-
minating in some kinds of oligosaccharides.7,8)
-
b
-
D
ª
1)-
b
-
-
D
-fructofurano-
-glucopyrano-
O
b
and
-
D
ª
4)]-
a
D
1F(1-
b-
D
-fructofuranosyl)2-4G-
b-D-galac-
O
-
-
b
D
-fructofuranosyl-(2
ª
ª
1)]2-
4)]-
-
D
-fructofuranosyl-
O
-[
b
D
-glucopyranoside, respectively. Both oligosaccharides
We now report the syntheses of new functional
oligosaccharides elongated with one or two addition-
al fructose units by fructosyl transfer from 1-kestose
were scarcely hydrolyzed by carbohydrase from rat
small intestine.
to 4G-
b- -galactopyranosylsucrose by using aspara-
D
Human intestinal bacterial growth by 1F-
b
-
D
-fruc-
-galactopyranosylsucrose was com-
pared with that by the tetrasaccharides, stachyose and
nystose. Biˆdobacteria utilized 1F-
-fructofuranosyl-
4G-
-galactopyranosylsucrose to the same extent as
tofuranosyl-4G-
b
-
D
gus 1F-FT,8) and then test utilization of the saccharide
synthesized by Biˆdobacteria and other intestinal
b-
D
bacteria.
b-
D
The synthesis of saccharides A and B from mixture
of 1-kestose and 4G-
b
- -galactopyranosylsucrose was
D
stachyose or nystose. On the other hand, the unfavora-
ble bacteria, Clostridium perfringens, Escherichia coli
and Enterococcus faecalis, that produce mutagenic sub-
stances did not use the synthetic oligosaccharide.
investigated by using asparagus 1F-FT puriˆed ac-
cording to the improved method reported in the
previous paper.8) A mixture of asparagus 1F-FT
(0.02 U), 0.1
M
1-kestose and 0.1
M
4G-
b-D-galac-
Key words: fructosyloligosaccharide;
indigestible
tosylsucrose in a McIlvaine buŠer (pH 5.5, 0.1 ml)
9
oligosaccharide; 1F-fructosyltransferase;
was incubated at 30 C for 0, 2, 5, 10, 20 and 48 h in
Biˆdobacterium
the presence of a small amount of toluene. The reac-
tion was terminated by heating in a boiling water
bath for 5 min, and the reaction mixture was subject-
ed to HPAEC.9–11) As shown in Fig. 1, saccharides A,
B and nystose accompanied with sucrose were pro-
We have recently investigated the enzymatic
production of non-digestible oligosaccharides having
activities as ``tertiary functional ingredients'' of
foods. Fructo-oligosaccharides synthesized from
sucrose by Eurotium repense fructosyltransferase1)
had no elevating eŠect on the blood glucose and insu-
lin concentrations in rats.1) Inulo-oligosaccharides
produced from inulin with Penicillium purpuroge-
num inulinase2,3) reduced the serum cholesterol level
in rats.1) Fructosylxyloside4) formed from sucrose and
xylose by the fructosyltransferase action of
Scopulariopsis brevicaulis cells suppressed the serum
duced from 1-kestose and 4G-
D
b- -galactopyranosyl-
sucrose with the 48-h reaction. The syntheses of sac-
charide A and nystose reached the maximum level of
9.7 mg and 6.5 mg per ml of the reaction mixture af-
ter 48 h and proceeded at a much higher rate than the
synthesis of saccharide B. A reaction mixture (25 ml,
0.5 U of enzyme) with the same components was in-
9
cubated at 30 C for 20 hours. After the reaction had
been terminated by heating in a boiling water bath
for 5 min, a 0.2-ml aliquot of the mixture was sub-
jected to repeated preparative HPLC equipped with
glucose and insulin responses5) and or promoted the
W
absorption of calcium and magnesium ions5) in rats
administered with sucrose. These oligosaccharides
also selectively stimulated the growth of Biˆdobac-
×
ODS column (Tosoh, TSKgel ODS-80Ts, 20 mm
25 cm). Each eluate corresponding to saccharide A
†
To whom correspondence should be addressed. Phone: +81-11-388-4754; Fax: +81-11-387-5848; E-mail: n-shiomi
＠
rakuno.ac.jp

1420
A. YAMAMORI et al.
Table 1. 13C-NMR Chemical Shifts of Saccharides A and B
Formed by Asparagus 1F-FT
Carbon atom
Saccharide A
Saccharide B
Terminal fructose
C-1
C-2
C-3
C-4
C-5
C-6
61.30
104.57
77.43
75.32
81.96
63.15
61.24
104.50
77.58
75.17
81.91
63.08
Middle fructose
C-1
C-2
C-3
C-4
C-5
C-6
61.74
103.88
78.34
75.29
81.91
63.08
Inner fructose
C-1
C-2
C-3
C-4
C-5
C-6
Glucose
C-1
C-2
C-3
C-4
C-5
C-6
Fig. 1. HPAEC of Saccharides Produced from 1-Kestose and 4G-
61.69
104.12
77.50
74.69
82.08
62.99
61.88
104.09
77.58
74.71
82.10
63.00
b-
D
-Galactopyranosylsucrose by Asparagus 1F-FT.
The enzymatic incubation was carried out for 48 h with 0.1
1-kestose and 4G-
-galactosylsucrose in the mixture. The reac-
tion mixture was diluted 100 times with distilled water, and an
M
b-
D
aliquot (25
tails).
ml) was subjected to HPAEC (see the text for de-
A, saccharide A; B, saccharide B; S, sucrose; GS, 4G-
topyranosylsucrose; 1-K, 1-kestose; Nys, nystose
b- -galac-
D
93.07
71.65a
72.08
78.93
71.92a
60.37
93.08
71.69b
72.08
78.93
71.94b
60.38
and saccharide B was collected, evaporated at 359C,
and rechromatographed in the same manner. The
two eluates were separately concentrated in vacuo
and lyophilized to give white powder of saccharide A
Galactose
C-1
C-2
C-3
C-4
C-5
C-6
103.75
71.83a
73.39
69.41
76.21
61.88
103.75
71.83b
73.39
69.41
76.21
61.88
(120 mg, 9.5
z
yield from the donor saccharide) and
saccharide B (19 mg, 1.5
charide).
z yield from the donor sac-
Saccharides A ([a]2D2+32.5) and B ([a]2D2+21.7)
Chemical shifts are expressed in ppm downˆeld from the signal for TMS,
relative to which the 1, 4 dioxane signal appears at 67.40. The assigments
of the resonances marked with a or b may be exchangeable.
were found to be homogeneous by PC12) (Rsucrose
,
d
0.230 and 0.160), TLC12) (Rsucrose, 0.330 and 0.266,
pre-coated plate), HPAEC (tRsucrose, 1.72 and 2.28)
and HPLC (Tosoh, TSKgel ODS-80Ts, 4
×
25 cm;
t
Rsucrose, 2.98 and 4.37). A and B were respectively
methyl-
D
-fructoside, methyl 2,3,6-tri-
O
-methyl-
D
-
-
found to be a tetrasaccharide and pentasaccharide
made up of fructose (A, 2 mol; B, 3 mol), glucose (A
and B, 1 mol) and galactose (A and B, 1 mol) by
glucoside and methyl 2,3,4,6-tetra-
O
-methyl-
D
galactoside. The two peaks corresponding to methyl
3,4,6-tri-
O
-methyl- -fructoside observed in the
D
measuring the [M+Na]+ ions (m z: A, 689 and B,
methanolysate of permethylated saccharide B were
larger than those peaks in that of permethylated sac-
charide A.
W
851) by TOF-MS and molar ratios (A, 2.1:1.0:1.0; B,
3.2:1.0:1.0) of
D
-fructose,
D
-glucose and
D
-galactose
in enzymatic hydrolysates (
b
-fructofuranosidase
b
-
Saccharides A and B were thus proved to be 1F-
b
D
-
-
＋
galactosidase) of the saccharides by HPLC equipped
with Sugar-Pak column (Ca-type, 7.8 mm
Nihon Waters K. K., Japan).
D
-fructosyl-4G-
fructosyl)2-4G-
b
-
D
-galactosylsucrose and 1F(1-
-galactosylsucrose.
b
-
×
30 cm,
b
-
D
The structures of saccharides A and B were con-
ˆrmed by a 13C-NMR analysis according to the data
shown in Table 1. The general assignment of
resonances in the spectrum of saccharide A in D2O
was tentatively made by comparing the observed
chemical shifts with the data for 1-kestose,14) nys-
To clarify the bond structures of the component
sugars, saccharides A and B were permethylated by
the Hakomori method.13) The permethylated saccha-
rides were methanolyzed with 1.5
z
methanolic
hydrogen chloride and subjected to GLC as described
in the previous paper.12)
tose14) and 4G-
b- -galactopyranosylsucrose.
D
The methanolyzate of permethylated saccharide A
gave seven peaks corresponding to methyl 1,3,4,6-
All of 13C-NMR data are in accord with the
clariˆed structures of saccharides A and B shown in
Fig. 2.
tetra-
O
-methyl- -fructoside, methyl 3,4,6-tri-O-
D

New Oligosaccharides Formed by Asparagus 1F-Fructosyltransferase
1421
Fig. 2. Structures of Saccharides A and B Formed by Asparagus 1F-FT.
Table 2. Utilization of SaccharideA and Several Other Saccharides by Some Human Intestinal Bacteria
Bacterial species
Saccharide A
Sta
Nys
LS
1-K
Suc
Lac
Glc
Biˆdobacterium adolescentis 2793
B. biˆdum 2777
B. breve 2776
B. infantis 2775
B. longum 2778
Lactobacillus acidophilus 2243
L. casei 2036
L. fermentum 2046
Enterobacter cloaceae 1180
Escherichia coli 1099
Enterococcus faecalis 2048
Clostridium perfringens 1211
Z
Z
Z
Z
Z
+
—
—
—
—
—
—
Z
Z
Z
Z
Z
—
—
—
—
—
—
—
Z
Z
Z
Z
Z
—
—
—
—
—
—
—
Z
Z
[
Z
Z
+
—
—
—
—
—
—
Z
Z
[
Z
Z
Z
—
—
—
—
—
—
Z
Z
[
[
Z
Z
Z
Z
+
Z
Z
±
Z
Z
Z
Z
Z
Z
Z
Z
+
Z
—
—
Z
Z
[
Z
[
Z
[
Z
+
Z
Z
±
Sta, stachyose; Nys, nystose; LS, 4G-
b- -galactopyranosylsucrose; 1-K, 1-kestose; Suc, sucrose; Lac, lactose; Glc, glucose
D
*
Saccharides A and B formed by fructosyl transfer
to 4G-
-galactopyranosylsucrose from 1-kestose
with asparagus 1F-FT were conˆrmed to be two new
1-Kestose and nystose were also not hydrolyzed by
the crude enzyme preparation. Therefore, saccha-
rides A and B were indigestible.
b
-
D
saccharides,
1F-
b
-
D
-fructofuranosyl-4G-
b
-
D
-galac-
1)-
4)]-
-fructofuranosyl)2-
-galactopyranosylsucrose, [ -fructofura-
1)]2- -fructofuranosyl- -galacto-
pyranosyl-(1 4)]- -glucopyranoside.
The utilization of saccharide A by human intesti-
nal bacteria was studied. Biˆdobacteria and Lac-
tobacilli are beneˆcial to both the nutrition and
health of humans and animals, while some intestinal
bacteria such as Escherichia coli, Enterobactor cloa-
ceae, Enterococcus faecalis and Clostridium perfrin-
gens are detrimental intestinal bacteria. The in vitro
utilization of several saccharides, especially tetrasac-
charides, by human intestinal bacteria is shown in T-
able 2. The bacterial growth was measured by analys-
topyranosylsucrose,
O
-
b
b
-
-
D
-fructofuranosyl-(2
ª
b-
D
-fructofuranosyl-
O
- [
D
-galactopyranosyl-(1ª
a
-
D
-glucopyranoside and 1F(1-
b
-
D
4G-
b
-
D
O
-
b
-
D
nosyl-(2
ª
b
-
D
O
-
[b
-
D
ª
a
-
D
The indigestibility of saccharides A and B was
examined by using rat intestinal disaccharidase.15)
Neither saccharide A nor B was hydrolyzed by the ac-
tion of
from rat small intestinal mucosa, although each sac-
charide was slightly degraded by the action of
a
-glucosidases in a crude enzyme preparation
ing the pH value of the medium.16) The symbols ``
[
±
'', ``
Z
º
'', ``+'', `` '' and ``„`` respectively show
b
-
pH
4.5, 4.5–5.0, 5.0–5.5, 5.5–6.0, 6.0 of the
À
galactosidase in the enzyme preparation to release a
small amount of galactose and 1-kestose or nystose.
medium. Biˆdobacterium adolescentis 2793, B. biˆ-
dum 2777, B. breve 2776, B. infantis 2775, B. lon-

1422
A. YAMAMORI et al.
nucleotide sequences of cDNA and gene encoding
endo-inulinase from Penicillium purpurogenum.
Biosci. Biotechnol. Biochem., 60, 1780–1785 (1996).
4) Takeda, H. and Kinoshita, S., Production of fruc-
tosyl-xylosides by Scopulariopsis brevicaulis sp. J.
Ferment. Bioeng., 79, 242–246 (1995).
5) Fukumori, Y., Onodera, S., and Shiomi, N., Nutri-
tional characteristics of substances related to pentose
and sugar alcohol. J. Appl. Glycosci., 48, 205–213
(2001).
gum 2778 utilized saccharide A to the same extent as
stachyose and nystose, which are respectively known
to be one of the main components of soy oligosac-
charide and fructooligosaccharide, although 4G-
D
b- -
galactopyranosylsucrose and 1-kestose were more
eŠectively utilized by B. breve
.
Under the normal conditions for Biˆdobacterium
growth, the pH value of the medium without saccha-
ride (control), and with saccharide A, stachyose, nys-
tose, 4G-
b-D-galactopyranosylsucrose, 1-kestose, lac-
6) Shiomi, N., Properties of fructosyltransferases in-
volved in the synthesis of fructan in Liliaceous plants.
J. Plant Physiol., 134, 151–155 (1989).
tose, sucrose or glucose added for Biˆdobacteria
growth was respectively 6.77–6.89, 4.59–4.66, 4.52–
4.63, 4.65–5.11,4.39–5.83, 4.49–4.83, 4.57–4.64,
4.44–4.66 and 4.48–4.65. On the other hand, neither
7) Shiomi, N., Onodera, S., and Sakai, H.,
Asparagosin-like fructan synthesized from 1-kestose
and neokestose by asparagus 1F-fructosyltransferase.
Abstracts of papers, Plant Polysaccharide Symposi-
um, Davis, USA, P30 (1998).
saccharide A nor stachyose, nystose, 4G-
b-D-galac-
topyranosylsucrose or 1-kestose was used by E. coli,
Enterobacter cloaceae, Enterococcus faecalis and C.
perfringens. Saccharide A was selectively used by ˆve
strains of the beneˆcial bacteria, Biˆdobacteria and
8) Shiomi, N., Puriˆcation and characterisation of 1F-
fructosyltransferase from the roots of asparagus
(
Asparagus o‹cinalis L.). Carbohydr. Res., 99,
Lactobacillus acidophilus
Saccharide A was more indigestible than 4G-
galactopyranosylsucrose and had lower osmotic
pressure in a solution than 4G-
-galactopyranosyl-
.
157–169 (1982).
b-D-
9) Rocklin, R. D. and Pohl, C. A., Determination of
carbohydrates by anion exchange chromatograpy
with pulsed amperometric detection. J. Liq. Chro-
matogr., 6, 1577–1590 (1983).
10) Johnson, D. C., Carbohydrate detection gains poten-
tial. Nature, 321, 451–452 (1986).
b
-
D
sucrose or 1-kestose, making it better for intestinal
conditions.
11) Shiomi, N., Onodera, S., Chatterton, N. J., and
Harrison, P. A., Separation of fructooligosaccharide
isomers by anion exchange chromatography. Agric.
Biol. Chem., 55, 1427–1428 (1991).
12) Shiomi, N., Yamada, J., and Izawa, M., Isolation
and identiˆcation of fructo-oligosaccharides in roots
of asparagus (Asparagus o‹cinalis L.). Agric. Biol.
Chem., 40, 567–575 (1976).
13) Hakomori, S., A rapid permethylation of glycolipid,
and polysaccharide catalyzed by methylsulˆnyl car-
banion in dimethyl sulfoxide. J. Biochem., 55,
208–208 (1964).
Acknowledgments
This work was supported in part by Grant-aid for
the promotion of high-technology centered projects
from the Ministry of Education of Japan (2000) for
which the authors express their appreciation. We
thank Dr. E. Fukushi (Hokkaido University,
Sapporo) for measuring the 13C-NMR spectra.
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