Transglucosylation activities of multiple forms of alpha-glucosidase from spinach.

Article abstract of DOI:10.1271/bbb.67.1160

Transglucosylation activities of spinach alpha-glucosidase I and IV, which have different substrate specificity for hydrolyzing activity, were investigated. In a maltose mixture, alpha-glucosidase I, which has high activity toward not only maltooligosaccharides but also soluble starch and can hydrolyze isomaltose, produced maltotriose, isomaltose, and panose, and alpha-glucosidase IV, which has high activity toward maltooligosaccharides but faint activity toward soluble starch and isomaltose, produced maltotriose, kojibiose, and 2,4-di-alpha-D-glucosyl-glucose. Transglucosylation to sucrose by alpha-glucosidase I and IV resulted in the production of theanderose and erlose, respectively, showing that spinach alpha-glucosidase I and IV are useful to synthesize the alpha-1,6-glucosylated and alpha-1,2- and 1,4-glucosylated products, respectively.

Full text of DOI:10.1271/bbb.67.1160

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Transglucosylation Activities of Multiple Forms of α-
Glucosidase from Spinach
a
ac
b
a
Manabu SUGIMOTO , Satoshi FURUI , Kenji SASAKI & Yukio SUZUKI
a
Research Institute for Bioresources, Okayama UniversityKurashiki, Okayama 710-0046,
Japan
b
Faculty of Pharmaceutical Sciences, Okayama UniversityOkayama, Okayama 700-8530,
Japan
c
Present address: Nippon Gene Co., Ltd.Toyama, Japan
Published online: 22 May 2014.
To cite this article: Manabu SUGIMOTO, Satoshi FURUI, Kenji SASAKI & Yukio SUZUKI (2003) Transglucosylation Activities
of Multiple Forms of α-Glucosidase from Spinach, Bioscience, Biotechnology, and Biochemistry, 67:5, 1160-1163, DOI:
10.1271/bbb.67.1160
To link to this article: http://dx.doi.org/10.1271/bbb.67.1160
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Biosci. Biotechnol. Biochem., 67 (5), 1160–1163, 2003
Note
Transglucosylation Activities of Multiple Forms of
a
-Glucosidase from Spinach
Manabu SUGIMOTO,1, Satoshi FURUI,1,* Kenji SASAKI,2 and Yukio SUZUKI
†
1
1
Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan
Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Okayama 700-8530, Japan
2
Received November 13, 2002; Accepted February 6, 2003
Transglucosylation activities of spinach a-glucosidase
I and IV, which have diŠerent substrate speciˆcity for
hydrolyzing activity, were investigated. In a maltose
mixture, a-glucosidase I, which has high activity toward
not only maltooligosaccharides but also soluble starch
and can hydrolyze isomaltose, produced maltotriose,
isomaltose, and panose, and
a-glucosidase IV, which
has high activity toward maltooligosaccharides but faint
activity toward soluble starch and isomaltose, produced
maltotriose, kojibiose, and 2,4-di-
Transglucosylation to sucrose by
resulted in the production of theanderose and erlose,
respectively, showing that spinach -glucosidase I and
IV are useful to synthesize the -1,6-glucosylated and
-1,2- and 1,4-glucosylated products, respectively.
a-
D
-glucosyl-glucose.
a-glucosidase I and IV
a
a
a
Key words:
a-glucosidase; spinach; transglucosylation
Transglycosylation by carbohydrases has been
used for the improvement of chemical and physiolog-
ical properties of various compounds. The
glycosylated compounds such as vitamins,
ribonucleosides, sugar alcohols, and glycosides
increased the solubility in water and stability against
1
)
heat or oxidation. Furthermore, biofunctional
oligosaccharides used for the prevention of disease
and maintenance of health are synthesized by using
Fig. 1. TLC Analysis of the Reaction Mixture.
The maltose solution was incubated for 8 h with spinach a-
glucosidases. Anisaldehyde solvent was used for sugar detec-
2
)
enzymatic transglycosylation.
.2.1.20] is an exo-carbohydrase, which catalyzes the
splitting of an -glucosyl residue from the nonreduc-
ing terminal of substrate to liberate -glucose, and
also catalyzes the transglucosylation to synthesize
a-Glucosidase [EC
tion. S, standard sugars; I,
IV.
a-glucosidase I; IV, a-glucosidase
3
a
a
a
-
high activity toward maltooligosaccharides but faint
activity toward soluble starch and isomaltose. In this
study we characterized the transglucosylation activi-
3
,4)
glucosylated compounds. The substrate speciˆcity
of the enzyme for hydrolyzing activity diŠers with
5
)
enzyme source and it is not expected that the
glucosylated hydroxyl group of the acceptor for
transglucosylation activity by )a-glucosidase depends
ties of spinach a-glucosidases I and IV, and used
them for the enzymatic synthesis of glucosyl sucrose,
which has the advantage of a property against
sucrose.
6
on its hydrolyzing speciˆcity.
We demonstrated that there are four molecular
The transglucosylation reaction was done in the
7
,8)
forms of
a
-glucosidase in spinach.
a
-Glucosidases
reaction mixture (40 ml) containing 10
z
maltose and
acetate buŠer (pH 5.0). The
mixture was incubated at 37 C and boiled for 10 min
to stop the reaction. Transglucosylated products
I and II have high activity toward not only mal-
tooligosaccharides but also soluble starch, and can
hydrolyze isomaltose. a-Glucosidases III and IV have
20 U of enzyme in 50 m
M
9
†
To whom correspondence should be addressed. Fax:
Present address: Nippon Gene Co., Ltd., Toyama, Japan
81-86-434-1249; E-mail: manabus rib.okayama-u.ac.jp
＋ ＠
*

Transglucosylation Activities of Spinach
a
-Glucosidases
1161
Fig. 2. Course of Transglucosylation in a Maltose Solution by Spinach
A reaction mixture containing 10 maltose and 20 U of enzyme in 50 m
, glucose; , maltotriose; , panose; , isomaltose; , kojibiose;
a
-Glucosidase I (A) and IV (B).
acetate buŠer (pH 5.0) was incubated at 37
, 2,4-di- -glucosyl-glucose.
z
M
9
C.

, maltose;

#
$



a-D
Fig. 3. Course of Transglucosylation to Sucrose by Spinach
A reaction mixture containing 10 sucrose, 10 soluble starch for
zyme in 50 m acetate buŠer (pH 5.0) was incubated at 37 C. Total composition of sugar except soluble starch and dextrin is shown as
00. , sucrose; , product by -glucosidase I which shows the same retention time as theanderose; , product by -glucosidase IV
a
-Glucosidase I (A) and IV (B).
z
z
a
-glucosidase I or dextrin for
a-glucosidase IV, and 20 U of en-
M
9
1


a
#
a
which shows the same retention time as erlose.
were separated by thin-layer chromatography (TLC)
using a HPTLC-Alufolien Kieselgel 60F256 plate
addition to maltose, which was released by maltose-
9)
hydrolyzing activity. This result shows that
glucosidase I has -1,6-transglucosyl activities. In a
transglucosylation by -glucosidase IV, the products
after 8 h of reaction were maltotriose, kojibiose, and
a-
(
Merk, Germany) and
a
solvent system of
a
acetonitrile-acetic acid-water (7:1:2, v v v). After
a
WW
development three times, the products were detected
by anisaldehyde reagent (Fig. 1) and measured by
1
0)
2,4-di-
were 10, 16, and 15
result shows that -glucosidase IV has not only a
a
-
D
-glucosyl-glucose, the contents of which
, respectively (Fig. 2B). This
-1,4-
-1, 2-trans-
densitometry. In a transglucosylation reaction by
a
-
z
glucosidase I, panose was produced rapidly and the
production reached an equilibrium by 1 h, whereas
isomaltose and maltotriose increased gradually.
After the reaction for 8 h, the contents of mal-
totriose, isomaltose, and panose were 2, 10, and
a
transglucosylation activity but also
glucosylation activity.
a
Glucosyl sucrose, xylooligosaccharides, and galac-
tooligosaccharides, which are used as less or non-
1
1)
2
2
z
, respectively (Fig. 2A). The synthesis of
cariogenic sugar substitutes
and increase the
disaccharides is due to glucose used as an acceptor in
number of Biˆdobacterium to improve constipation

1
162
M. SUGIMOTO et al
.
1
3
Fig. 4.
C-NMR Spectra of Glucosyl-sucrose Produced by Spinach
a
-Glucosidase I and IV in D
2
O.
(
A) product by -glucosidase I; (B) authentic theanderose; (C) product by a
a
-glucosidase IV; (D) authentic erlose.
1
2–14)
through improvement of fecal conditions,
are
were the same as those of theanderose and erlose,
respectively (Fig. 4). These results indicate that the
production of theanderose and erlose is due to the
produced by enzymatic transformation from su-
2
,15,16)
crose.
Sucrose is a poor substrate for hydrolyz-
7)
ing by spinach
a
-glucosidases and these enzymes
a-1,6- and 1,4-transglucosylation activities of a-
would be suitable to synthesize glucosyl sucroses by
their transglucosylation. To verify the 1,4-trans-
glucosidase I and IV, respectively.
In this work, we showed that spinach
I and IV are useful to synthesize the
ated and
respectively.
a
-glucosidase
-1,6-glucosyl-
a-1,2- and 1,4-glucosylated products,
glucosylation activity of
a
-glucosidase I and 1,2-
-glucosidase IV
a
transglucosylation activity of the
a
and apply it to the production of glucosyl sucrose,
transglucosylation to sucrose was done in the reac-
tion mixture (40 ml) containing 10
soluble starch for -glucosidase I or dextrin (pine-dex
1 DE 8 1, Hayashibara Biochem. Lab. Inc.,
Japan) for -glucosidase IV, and 20 U of enzyme in
0 m acetate buŠer (pH 5.0). The mixture was incu-
bated at 37 C for 4 h and boiled for 10 min to stop
z
sucrose, 10
z
Acknowledgments
a
#
±
We wish to thank Dr. S. Chiba, Hokkaido Univer-
sity, for his invaluable advice on the analysis of
oligosaccharides. This research was supported in part
by the Ohara Foundation in Kurashiki.
a
5
M
9
the reaction because the main products produced by
a
-glucosidase I and IV, which showed the same reten-
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