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which the xylose unit was positioned at the nonreducing end of the
xylosylated oligosaccharides. Consequently, the treatment of the
reaction mixture with glucoamylase (GA, EC 3.2.1.3), which catalyzed
an exo-wise hydrolysis at the nonreducing end of
4)-glucans,
to reveal whether the xylose unit was positioned at the nonreducing
end. If the xylose unit is positioned at the nonreducing end of a
maltooligosaccharide, GA does not recognize it as a substrate. The
1
α
-(1
→
H NMR spectrum of the treated products indicated the remaining
signals due to the xylosidic linkages, supporting that the xylose
unit was positioned at the nonreducing end. Furthermore, the main
product was isolated from the crude mixture using high performance
liquid chromatography (HPLC) and the structure was confirmed by
the
1
. For further
analysis, the formation of the xylosylated maltooligosaccharides vs.
reaction time in the phosphorylase-catalyzed
H NMR spectrum to be
α
-
d
-xylosyl-(1
→
4)-Glc
4
-xylosylation was
evaluated under the conditions as the feed molar ratios of Glc
α
to Xyl-
1-P as 1:10, 1:5, and 1:1. The total yields of the xylosylated products
were estimated by the integrated ratios in the
4
1
H NMR spectra of the
crude products. Highest yields of 44% (1:10), 25% (1:5), and 10%
(1:1) based on the amounts of Glc
used were obtained after 48 h.
4
Schwarz
. reported the kinetic analysis using Glc-1-P and Xyl-1-P
in the phosphorylase-catalyzed reaction. Phosphorylase displayed
a very large kinetic selectivity ratio, i.e.,
et al
k
/
k
= 2000
catGlc-1-P
catXyl-1-P
[33].
-mannose
1-phosphate (Man-1-P) was also examined (Fig. 3.11) [34]. Although
the equatorial hydroxy group is not essential for phosphorylase,
2-epimer of Glc-1-P, i.e., Man-1-P, is expected to have a low affinity
to the enzyme owing to steric hindrance by the axial hydroxy group.
In the crude products obtained by the phosphorylase-catalyzed
reaction of Man-1-P with Glc
The phosphorylase-catalyzed
α
-mannosylation using
α
, mannosylated compounds were
found. A pentasaccharide fraction was isolated by size exclusion
chromatography and the structures were confirmed by the
4
1
H NMR
. The
mixture was not separated completely. Particularly, the position of
the mannose unit was assigned owing to the lower intensity of the
terminal H-4 signal in comparison with the integral of
spectrum to be a mixture of Glc
and
α
-mannosyl-(1
→
4)-Glc
5
4
α
- and
β
-H-1
at the reducing end.
