Three novel oligosaccharides synthesized using Thermoanaerobacter brockii kojibiose phosphorylase
Chemistry Central Journal
Three novel oligosaccharides synthesized using Thermoanaerobacter brockii kojibiose phosphorylase
Natsuko Takahashi 2
Eri Fukushi 1
Shuichi Onodera 2
Noureddine Benkeblia 2
Tomoyuki Nishimoto 0
Jun Kawabata 1
Norio Shiomi 2
0 Hayashibara Biochemical Laboratories, Inc. , Okayama 702-8006 , Japan
1 Graduate School of Agriculture, Hokkaido University , Sapporo 060-8589 , Japan
2 Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University , Ebetsu 069-8501 , Japan
Background: Recently synthesized novel oligosaccharides have been produced primarily by hydrolases and glycosyltransferases, while phosphorylases have also been subject of few studies. Indeed, phosphorylases are expected to give good results via their reversible reaction. The purpose of this study was to synthesis other novel oligosaccharides using kojibiose phosphorylase. Results: Three novel oligosaccharides were synthesized by glucosyltransfer from β-D-glucose 1phosphate (β-D-G1P) to xylosylfructoside [O-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside] using Thermoanaerobacter brockii kojibiose phosphorylase. These oligosaccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methyl derivatives, MALDI-TOF MS and NMR measurements were used for structural characterisation. The 1H and 13C NMR signals of each saccharide were assigned using 2D-NMR including COSY (correlated spectroscopy), HSQC (herteronuclear single quantum coherence), CH2-selected E-HSQC (CH2-selected Editing-HSQC), HSQC-TOCSY (HSQC-total correlation spectroscopy) and HMBC (heteronuclear multiple bond correlation). Conclusion: The structure of three synthesized saccharides were determined, and these oligosaccharides have been identified as O-α-D-glucopyranosyl-(1→2)-O-α-D-xylopyranosyl(1→2)-β-D-fructofuranoside (saccharide 1), O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl(1→2)-O-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside (saccharide 2) and O-α-Dglucopyranosyl-(1→[2-O-α-D-glucopyranosyl-1]2→2)-O-α-D-xylopyranosyl-(1→2)-β-Dfructofuranoside (saccharide 3).
Background
The synthesis of oligosaccharides with various functions
has been actively performed for some time. Such
oligosaccharides are primarily synthesized by hydrolases and
glycosyltransferases. Although phosphorylases have been the
subject of few studies, they are expected to give good
results via their reversible reaction.
Thermoanaerobacter brockii kojibiose phosphorylase is
known to catalyze the reversible phosphorolysis of
kojibiose into β-D-glucose 1-phosphate (β-D-G1P) and glucose
[
1
]. In this reversible reaction, the enzyme uses various
oligosaccharides as accepters. Furthermore, this enzyme is
understood to catalyze glucosyltransfer from β-D-G1P to
position 2 of the glucose residue in oligosaccharides such
as raffinose, stachyose [
2
], 1-kestose, nystose [
3
] and
sucrose.
Xylosylfructoside
[O-α-D-xylopyranosyl-(1→2)-β-Dfructofuranoside] is an oligosaccharide that exhibits
noncariogenicity and selective growth stimulation in
bifidobacteria. This oligosaccharide does not exist naturally, but
can be synthesized by levansucrases from Bacillus subtilis
[
4
] and Aerobacter levanicus [
5
] or β-fructofuranosidases
from Penicillum sp. K-25 [
6
], Penicillum frequertans T-1[
7
]
and Scopulariopsis brevicaulis [
8
].
In this paper we report when xylosylfructoside is used as a
substrate, Thermoanaerobacter brockii kojibiose
phosphorylase catalyzes glucosyltransfer from β-D-G1P to position 2
of the xylose residue. However transfer to other
saccharides lacking glucose residues does not occur, with the
exception of sorbose.
Here, we succeed in the synthesis of three new
oligosaccharides, saccharides 1, 2 and 3, by glucosyltansfer from
β-D-G1P to xylosylfructoside using Thermoanaerobacter
brockii kojibiose phosphorylase.
We also carried out structural analysis of the synthesized
oligosaccharides using NMR spectroscopy. Structural
analysis using NMR of the saccharides with a high degree
of polymerization by NMR is now becoming a standard
technique. However, it is difficult to assign the proton
(1H) and carbon (13C) signals in oligosaccharides whose
residues are similar, particularly in oligosaccharides with
numerous methylene (CH2) groups, such as
fructooligosaccharides and kojioligosaccharides.
The purpose of this study is to synthesize three novel
oligosaccharides by kojibiose phosphorylase and carry out
the full assignment of the 1H and 13C signals using
2DNMR techniques such as COSY, HSQC, CH2 E-HSQC,
HSQC-TOCSY and HMBC.
Results and discussion
Oligosaccharide synthesis and identification
Saccharides 1, 2 and 3 were synthesized from
xylosylfructoside
[O-α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside] and β-D-G1P using kojibiose phosphorylase. The
HPAEC chart of saccharides 1, 2 and 3 synthesized after
54 h reaction is sh (...truncated)