Purification and characterization of thermostable α-galactosidase from Ganoderma lucidum

Article abstract of DOI:10.1271/bbb.67.1485

α-Galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea α-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70°C, respectively. The enzyme was fully stable to heating at 70°C for 30 min. It hydrolyzed p-nitrophenyl-α-D- galactopyranoside (K_m = 0.4 mM) but hydrolyzed little o-nitrophenyl-α-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses.

Full text of DOI:10.1271/bbb.67.1485

This article was downloaded by: [York University Libraries]
On: 12 August 2014, At: 09:17
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Bioscience, Biotechnology, and Biochemistry
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tbbb20
Purification and Characterization of Thermostable α-
Galactosidase from Ganoderma lucidum
Thida SRIPUANab, Kazuhiro AOKIc, Kenji YAMAMOTOc, Dararat TONGKAOa & Hidehiko
KUMAGAIc
a Department of Chemistry, Faculty of Science, Chiang Mai UniversityChiang Mai 50200,
Thailand
b Institute for Science and Technology Research and Development, Chiang Mai
UniversityChiang Mai 50200, Thailand
c Division of Integrated Life Science, Graduate School of Biostudies, Kyoto
UniversityKyoto, Japan
Published online: 22 May 2014.
To cite this article: Thida SRIPUAN, Kazuhiro AOKI, Kenji YAMAMOTO, Dararat TONGKAO & Hidehiko KUMAGAI (2003)
Purification and Characterization of Thermostable α-Galactosidase from Ganoderma lucidum , Bioscience, Biotechnology,
and Biochemistry, 67:7, 1485-1491, DOI: 10.1271/bbb.67.1485
To link to this article: http://dx.doi.org/10.1271/bbb.67.1485
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Biosci. Biotechnol. Biochem., 67 (7), 1485–1491, 2003
Puriˆcation and Characterization of Thermostable
a
-Galactosidase from
Ganoderma lucidum
, ,
† Kazuhiro AOKI,3 Kenji YAMAMOTO,3 Dararat TONGKAO
1,2,
1
Thida SRIPUAN
3
and Hidehiko KUMAGAI
1Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
2Institute for Science and Technology Research and Development, Chiang Mai University,
Chiang Mai 50200, Thailand
3Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
Received January 9, 2003; Accepted March 17, 2003
a
-Galactosidase was puriˆed from a fresh fruiting
Previous studies of G. lucidum have mainly con-
centrated on the medicinal properties of this
mushroom. Limited information is available on the
activities of the enzymes which modify oligo- and
polysaccharides present in the mushroom, though
preliminary reports4–7) have shown that G. lucidum
used galactose as the best carbon source for produc-
ing potentialized ganoderan having anti-tumor and
anti-complementary activity. We found that cultivat-
ed G. lucidum produced a wide variety of glycosi-
body of Ganoderma lucidum by precipitation with am-
monium sulfate and column chromatographies with
DEAE-Sephadex and Con A-Sepharose. The puriˆed
enzyme was homogeneous on polyacrylamide gel elec-
trophoresis. Its N-terminal amino acid sequence was
similar to that of Mortierella vinacea
a-galactosidase.
The molecular mass of the enzyme was about 56 kDa by
SDS-polyacrylamide gel electrophoresis, and about
249 kDa by gel ˆltration column chromatography. The
optimum pH and temperature were 6.0 and 70
respectively. The enzyme was fully stable to heating at
70 C for 30 min. It hydrolyzed -nitrophenyl-
galactopyranoside (Km 0.4 m ) but hydrolyzed little
-nitrophenyl- -galactopyranoside. It also hydro-
9C,
dases, and had strong
-Galactosidase (EC 3.2.1.22) is known to catalyze
the hydrolysis of the terminal -linked galactoside
residues from -galactosides including galacto-
a-galactosidase activity.
a
9
p
a-
D
-
a
＝
M
a-O
o
a-
D
containing polysaccharides such as galactomannans
lyzed melibiose, ra‹nose, and stachyose. The enzyme
catalyzed the transgalactosylation reaction which
synthesized melibiose. The product was conˆrmed by
various analyses.
and galacto(gluco)mannans.8,9) Despite the hydrolytic
activity, a-galactosidase is powerful for oligosaccha-
ride synthesis by transglycosylation, and also by
reverse hydrolysis.10–13) Therefore, the mechanism
for producing polysaccharides of G. lucidum might
be related to both the hydrolytic activity and galac-
Key words:
a-galactosidase; Ganoderma lucidum;
thermostable enzyme; transglycosylation;
galactooligosaccharide
tosyl transfer activity of
a
-galactosidase. We isolated
-galactosidase and
and characterized G. lucidum
a
found that this enzyme was relatively thermally sta-
ble and had the transfer activity for oligosaccharide
synthesis.
Ganoderma lucidum is a mushroom traditionally
used in oriental medicine because of its complex
polysaccharides which are known to stimulate the
immune response. Several polysaccharides obtained
from fruiting bodies of G. lucidum were reported to
have anti-tumor activity and consist of glucose,
galactose, mannose, and fucose.1,2) This mushroom is
known as the producer of ganoderan C, which is a
peptideglycan with hypoglycemic potency. Its glycan
Materials and Methods
Enzyme source. Ganoderma lucidum was collected
at the campus of Chiang Mai University. The fruiting
bodies of the mushrooms were used as a source of the
enzyme.
is composed of
D
-glucose (69.6
z
of peptideglycan)
and -galactose (2.9
D
z
of peptideglycan), and phys-
Preparation of crude extract of mushroom. The
mushroom fruiting bodies were extracted using
ico-chemical and chemical studies demonstrated that
the backbone and side chains of ganoderan C con-
100 m
M
sodium phosphate buŠer (pH 6.0), mixed in
tained
and a
D
-glucopyranosyl
b
-1
ª
3 and
b
-1
ª
6 linkages
a blender for 1 min and then ˆltered through cotton
D
-galactopyranosyl
a
-1
ª
6 linkage.3)
cloth. The ˆltrate was centrifuged at 7,500
g for
×
†
To whom correspondence should be addressed. Fax:
66-53-892188; E-mail: thidach yahoo.com
＋ ＠

1486
T. SRIPUAN et al.
15 min at 4
9
C. The supernatant obtained was used as
Molecular mass measurement. The molecular mass
of the puriˆed enzyme was estimated by gel ˆltration
with a Superdex 200HR column (Pharmacia-LKB).
The column was calibrated with high molecular
weight markers (Pharmacia-LKB). The column was
the starting crude enzyme preparation for puriˆca-
tion.
Enzyme assay. The activity of
assayed by measuring the amount of
released from -nitrophenyl NP)
topyranoside as a substrate. The standard reaction
mixture contained 4 m NP- -galactopyrano-
side, 200 m sodium phosphate buŠer (pH 6.0), and
the enzyme in a ˆnal volume of 0.6 ml. After incuba-
tion at 40 C for 10 min, 2.7 ml of 200 m borate
a
-galactosidase was
-nitrophenol
-galac-
p
pre-equilibrated with 50 m
M
sodium phosphate
NaCl, and eluted
p
(
p
a
-
D
buŠer (pH 7.0) containing 0.15
M
with the same buŠer at a ‰ow rate of 0.5 ml min.
W
M
p
a
-
D
M
Polyacrylamide gel electrophoresis. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
9
M
(SDS-PAGE) was done in a 12
pH 8.8 using 25 m
0.1
(w v) SDS as described by Laemmli.15) A low
z
acrylamide gel at
buŠer (pH 10.0) was added to the mixture to stop the
reaction. Thereafter, the absorbance of the mixture
was measured at 400 nm.
One unit of
amount of enzyme which released 1
nitrophenol from NP- -galactopyranoside per
M
Tris-glycine buŠer containing
z
W
molecular mass calibration mixture (Pharmacia-
LKB) was used as the standard marker. The gel was
stained with Coomassie Brilliant Blue R-250 (Nacalai
Tesque).
a
-galactosidase was deˆned as the
m
mol of
p-
p
a
-
D
min under the standard assay conditions. The speciˆc
activity was expressed as units per mg protein.
Native PAGE was done at 4
linear gradient slab gel with the running buŠer
(pH 8.4) consisting of 90 m Tris-HCl, 80 m
borate, and 2.5 m EDTA by the method of Manabe
Protein bands in the gel were stained with a
silver stain kit (Wako Chemicals). The activity stain-
ing of -galactosidase on the gel was done using 4-
methylumbelliferyl- -galactoside as substrate and
9C using a 4 to 30z
M
M
Protein measurement. The protein concentration
was measured by the method of Bradford using
bovine serum albumin as a standard14) or by measur-
ing the optical density at 280 nm. The latter method
was used for monitoring protein on column chro-
matographies.
M
16)
et al
.
a
a
-D
the activity band was made visible under UV light at
365 nm.
Puriˆcation of the enzyme. All puriˆcation proce-
dures were done at 4
9
C. Unless otherwise indicated,
N-Terminal amino acid sequence analysis. Puriˆed
10 m
M
potassium phosphate buŠer (pH 6.0) was used
enzyme (140 mg) was put through SDS-PAGE and
throughout the puriˆcation.
electroblotted onto an Immobilon-PSQ 0.45
vinylidene ‰uoride membrane (Millipore). The mem-
brane was stained with 0.025 Coomassie Brilliant
Blue R-250 in 40 methanol and washed with 50
mm poly-
To the crude extract (160 ml), solid ammonium
sulfate was added to the ˆnal 80
centrifugation (12,500
z
saturation. After
z
×
g
) for 30 min, the precipitate
z
z
was dissolved in the buŠer and dialyzed against the
same buŠer for 12 h. The dialyzed solution was put
methanol. The protein band was then cut out. The
strip of membrane holding the protein was put
through automatic Edman degradation for sequenc-
ing of the N-terminal amino acids using an Applied
Biosystems 477A protein sequencer (Perkin-Elmer).
to a column of DEAE-Sephadex A-50 (2.2
×
36 cm)
previously equilibrated with the buŠer. A stepwise
elution was done by increasing the NaCl concentra-
tion in the buŠer from 0 to 0.4
dase was mainly eluted with the buŠer containing
0.2 NaCl. The active fractions were pooled, con-
M
. The a-galactosi-
Kinetic studies. Kinetic studies were done under
M
various concentrations of structures such as
p
-
-
centrated by ultraˆltration, and dialyzed against the
same buŠer. Next, the dialyzed solution was put on a
nitrophenyl-
a
-
D
-galactopyranoside,
o
-nitrophenyl-
a
D
-galactopyranoside and
m
-nitrophenyl-
a
-
D
-galac-
×
column of Con A-Sepharose (1.2 5 cm) previously
equilibrated with 20 m Tris-HCl buŠer (pH 7.4)
containing 1 m each of MnCl2, MgCl2, and CaCl2,
and 0.5 NaCl. A stepwise elution was done by in-
creasing the concentration of methyl -mannoside
in 20 m Tris-HCl buŠer (pH 7.4) containing 0.5
NaCl from 0 to 1.0 . The -galactosidase was eluted
with the above buŠer containing 0.2 methyl
topyranoside. The apparent Michaelis constant (Km)
M
was calculated from the Lineweaver-Burk plot.
M
M
Thin layer chromatography (TLC). TLC was done
a
-
D
on a Kiesel gel 60 alumina plate (Merck) with a sol-
M
M
vent of n-propanol and water (80:20, v v). The sugar
W
M
a
compounds were visible after spraying with 0.2
orcinol in 1 sulfuric acid, followed by heating at
140 C.
z
M
a
-
D
-
M
mannoside. The active fractions were pooled, con-
centrated by ultraˆltration, and dialyzed against
9
10 m
M
potassium phosphate buŠer (pH 6.0). The
Sugar analyses. For the measurement of
tose released from natural oligosaccharides by the
enzyme, the reaction mixture containing 200 l of
D
-galac-
concentrated enzyme solution was used as the puri-
ˆed enzyme.
m

Thermostable
a
-Galactosidase from Ganoderma lucidum
1487
4 m
and stachyose), 200
buŠer (pH 6.0), and 200
M
natural oligosaccharides (melibiose, ra‹nose,
l of 200 m sodium phosphate
l of the enzyme solution
C for 10 min. After boiling for
-galactose was measured using
on DEAE-Sephadex A-50 and Con A-Sepharose.
The results of the enzyme puriˆcation are summa-
rized in Table 2. The enzyme was puriˆed about 12-
m
M
m
was incubated at 40
9
fold with a yield of 1.7z of total activity.
10 min, the released
D
a lactose
D
-galactose kit (Boehringer Mannheim
Homogeneity and molecular mass of the enzyme
The ˆnal enzyme preparation appeared to be
homogeneous on native PAGE (Fig. 1A) and stain-
ing using 4-methylumbelliferyl-a-galactopyranoside
(Fig. 1B) and presented as one major band on SDS-
PAGE (data not shown).
W
GmbH Biochemicals) by the UV method at 340 nm.
To examine the sugar composition of the com-
pound, TMS (trimethylsilyl) derivatives were put
through gas chromatography (GC-18A, Shimadzu,
Japan) with a capillary column of HiCap-CBP5
(25 m; Shimadzu, Japan), programmed at 2
9
C min
The N-terminal amino acid sequence of the puri-
ˆed enzyme was identiˆed as SNWGLAITPQ. To
W
from 150 to 230 C. To analyze the linkage of sugars,
9
permethylation was done ˆrst, according to the
method of Ciucanu and Kerek.17) The peaks on the
gas chromatogram were identiˆed by mass spectro-
metry (GCMS-QP 5050, Shimadzu, Japan) and by
comparison of the retention times with those of
partially methylated alditol acetates prepared from
authentic standards as a reference.
compare the amino acid sequence of the a-galactosi-
dase with the deduced amino acid sequences of other
genes, the FASTA database18) was searched. The N-
terminal amino acid sequence of G. lucidum a-galac-
tosidase was found to have the highest level of simi-
larity with the sequence (SNNGLAITPQ) between
positions 21 and 30 from the N-terminus of
tosidase from Mortierella vinacea
a-galac-
.
Chemicals. p-Nitrophenyl-
-nitrophenyl- -galactopyranoside,
phenyl- -galactopyranoside, and other glycosides
a
-
D
-galactopyranoside,
The molecular mass of the enzyme was estimated
to be about 56 kDa by SDS-PAGE and about
249 kDa by gel ˆltration column chromatography.
According to these results, the puriˆed a-galactosi-
dase is composed of 4 subunits each having a molecu-
lar mass of 56 kDa.
o
a
-
D
m
-nitro-
a
-
D
were purchased from Sigma Chemical Co. DEAE-
Sephadex A-50 and Con A-Sepharose were products
of Pharmacia.
a-Galactossidase of green coŠee bean
and -galactosidase of jack bean were obtained from
b
Sigma Chemical Co. and Seikagaku Kogyo Co., re-
spectively. All other reagents used were of the highest
purity available from commercial sources.
Table 1. Various Glycosidase Activities in the Extract of Fruiting
Body of Ganoderma lucidum
Results
Hydrolyzing activity
Glycosidase
(UWmg)
Glycosidase activities in the extract of fruiting bo-
dies of Ganoderma lucidum
a
a
a
a
a
a
a
a
a
a
-
-
-
-
-
-
-
-
-
-
D
D
D
D
D
D
D
D
D
D
-glucosidase
-glucosidase
-galactosidase
-galactosidase
-mannosidase
-mannosidase
-fucosidase
-fucosidase
-arabinosidase
-xylosidase
9
188
400
12
8
12
1
5
0
15
810
815
The glycosidase activities in the extract of
mushroom fruiting bodies were examined using
various
Table 1, various glycosidase activities were found.
Among them, the -galactosidase activity was stron-
p-NP-glycosides as substrates. As shown in
a
ger than the other activities except for
osaminidase activity.
b-N-acetylhex-
N
N
-acetyl-
-acetyl-
b
-
-
D
-glucosaminidase
b
D-galactosaminidase
Puriˆcation of
fruiting bodies of Ganoderma lucidum
The -galactosidase was puriˆed by ammonium
sulfate precipitation and column chromatographies
a-galactosidase from the extract of
The enzyme activities were measured at pH 6.0 using various
sides (4 m ) as substrates. The reaction was done at 40 C and the released
-nitrophenol was measured.
p-NP-glyco-
a
M
9
p
Table 2. Puriˆcation of the a-Galactosidase from Fruiting Body of Ganoderma lucidum
Total activity
(units)
Total protein
(mg)
Speciˆc activity
(units mg)
Yield
Puriˆcation steps
Crude extract
Ammonium sulfate precipitation (80
DEAE-Sephadex A-50 chromatography
Con A-Sepharose chromatography
W
z
( )
68.2
33.2
15.0
1.16
36.5
19.8
1.8
1.9
1.7
8.4
100
z
)
48.7
22.0
1.7
0.05
23.2

1488
T. SRIPUAN et al.
Fig. 1. Native Gradient Polyacrylamide Gel Electrophoresis and
the Activity Staining of Ganoderma lucidum -Galactosidase.
(A) The native PAGE was done using 4 to 30 acrylamide.
Lane 1: molecular size marker, Lane 2: the puriˆed -galactosi-
dase. (B) Activity staining of the puriˆed -galactosidase was
done using 4-methylumbelliferyl- -galactopyranoside.
Fig. 2. EŠects of Temperature on the Activity (A) and Stability
(B) of -Galactosidase.
The optimum temperature of the enzyme ( ) was measured
at pH 6.0 at various temperatures (20 to 100 C). The thermal
stability of the enzyme ( ) was measured by assaying the
remaining activity under standard conditions after incubating
the enzyme at pH 6.0 at various temperatures for 1 h.
a
a
z

9
a
a

a
Various properties of the enzyme
The optimum pH for enzymic activity was meas-
ured over a pH range from 1.2 to 8.0 using either
0.1
M
glycine buŠer (pH 1.2 to 3.2) or 0.1
M
McIlvaine buŠer (pH 3.2 to 8.0) under standard
assay conditions. The enzyme was most active at
pH 6.0.
The pH stability of the enzyme was measured by
the residual activity after incubation in 0.1
McIlvaine buŠer (pH 3.2 to 8.0) for 1 h at 40
enzyme was stable at pH 5.0 to 7.0.
M
9C. The
The optimum temperature for enzymic activity was
measured by a standard assay at various tempera-
9
tures ranging from 20 to 100 C and the thermal
stability of the enzyme was measured by assaying
residual activity after incubation of the enzyme at 20
Fig. 3. EŠects of Heat Treatment on the Stability of
a
-Galactosi-
dase.
The enzyme was heated at 70 C, 80 C, and 100 C for the
9 9 9
to 100
most active at 60 to 70
The eŠect of heat treatment on the enzyme was inves-
tigated by heating the enzyme at 70, 80, and 100
9
C for 1 h. As shown in Fig. 2, the enzyme was
indicated time, respectively, and the remaining activity was
assayed under standard conditions.
9
C and was stable below 70 C.
9
9
C
topyranoside was slightly susceptible, though the
for 30 min and the remaining activity was assayed.
As shown in Fig. 3, the enzyme was fully stable at
enzyme can scarcely act on
m
-NP-
a- -galac-
D
topyranoside. Natural substrates, such as melibiose,
ra‹nose, and stachyose, were good substrates for
70
9
C and about 30
z
of the original activity
C for 30 min.
remained after heating at 80
9
this enzyme. On the other hand,
glucopyranoside, -NP- -acetyl- -galactopyrano-
side, and -NP- -galactopyranoside were not
hydrolyzed by the enzyme. The Km values of the
enzyme for -NP- -galactopyranoside, -NP-
galactopyranoside, and -NP- -galactopyrano-
, and 16.7 m , respec-
p
-NP-
a
-
D
-
p
b
N
a
-
D
Substrate speciˆcity of the enzyme
p
-D
Synthetic substrates of glycosides and natural
oligosaccharides were used to investigate the sub-
strate speciˆcity of the enzyme. As shown in Table 3,
p
a
-
D
o
D
a- -
m
M
a
-D
p
-NP-
a
-
D
-galactopyranoside was the most suscepti-
-NP- -galac-
side were 0.4 m
tively.
M
, 0.67 m
M
ble substrate for this enzyme and
o
a
-D

Thermostable
a
-Galactosidase from Ganoderma lucidum
1489
Table 3. Substrate Speciˆcity of
ma lucidum
a-Galactosidase from Ganoder-
Substrate
z
Hydrolytic activity ( )
p
o
m
p
p
p
p
p
p
p
NP-
NP-
NP-
NP-
NP-
NP-
NP-
NP-
NP-
NP-
a
-
-
-
D
-galactoside
100.0
27.6
0.3
0.3
0
0.1
0.1
0
a
D
-galactoside
-galactoside
a
D
a-D-glucoside
N
N
-acetyl-
-acetyl-
a
a
-
-
D
-galactosaminide
-glucosaminide
D
b
-
D
-galactoside
-glucoside
b-
D
N
N
-acetyl-
-acetyl-
b
b
-
-
D
-galactosaminide
-glucosamonide
0
0
D
Melibiose
Ra‹nose
Stachyose
42.1
52.4
69.7
The enzyme activities were measured at pH 6.0 using various nitrophenyl-
glycosides (4 m ) or oligosaccharides (4 m ) as substrates. The reaction was
done at 40 C and released nitrophenol or galactose was measured by a
M
M
9
colorimetric method.
EŠects of various compounds and metal ions on
the enzymic activity
Fig. 4. Thin Layer Chromatography of the Transgalactosylation
The eŠects of various metal ions (1 m
M
) and other
Reaction Mixture.
reagents (1 m ) on the activity of the enzyme were
M
The transgalactosylation reaction was done using
galactopyranoside as the glycoside donor and glucose as the
acceptor. The reaction was done at 40 C for 24 h.
pNP-a-D-
examined under the standard enzyme assay condi-
tions. The enzyme activity intensely decreased to
9
32
z z
and 9 of the original activity by addition of
Ag＋ and Hg2＋, respectively. Other metal ions such as
Al3＋, Ba2＋, Ca2＋, Mn2＋, Zn2＋, K＋, and Na＋ and
Additionally, the reaction mixture with galactose as
an acceptor also showed the transgalactosylation
product on TLC, which was found in the same posi-
tion as the authentic disaccharide, melibose (data not
shown).
reagents such as b-mercaptoethanol and EDTA did
not aŠect the activity. The enzyme activity seemed to
＋
be slightly stimulated by Cu2 (about 1.2-fold).
The eŠects of addition of various sugars (10 m )
M
on the enzymic activity were also examined. The
activity was inhibited completely by galactose and
Analysis of the transgalactosylation product
slightly by xylose (88
z
of the original activity). Lac-
To isolate the transgalactosylation product (Gal-
tose, maltose, and sucrose did not aŠect the enzymic
activity.
Glc), the reaction mixture was put on a column (1
×
76 cm) of Iatrobeads 6RS-8060 (Iatron Lab. Inc.,
Tokyo, Japan) equilibrated with chloroform-
Transglycosylation activity of
a
-galactosidase
methanol-water at a ratio of 60:30:7 (v v). The frac-
W
The enzyme was found to have transglycosylation
activity in addition to hydrolyzing activity. The
tions containing the transgalactosylation product was
detected with phenol-H2SO4, pooled, and evaporat-
ed. The puriˆed product was methanolyzed, and the
methanolysates were converted to trimethylsilyl
(TMS) derivatives for analysis by gas chro-
matography (GC). The gas chromatograms of the
product deˆned the sugar component, of which
appropriate molar ratios were found after correction
for their relative molar responses as glucose and
galactose (molar ratio, 1:1) as shown in Fig. 5A. Sub-
sequently, to identify the sugar linkage, partially
methylated alditol acetate derivatives of the trans-
galactosylation product were prepared and analyzed
transglycosylation reaction was done in a 200 m
M
sodium phosphate buŠer (pH 6.0) in the presence of
0.1 unit of
a
-galactosidase per ml, 25 m
M
p
NP-
a
-
D
-
galactopyranoside as a glycoside donor, and 50 m
M
glucose as an acceptor. The incubation was done at
40 C for 24 hr, and the reaction was stopped by boil-
9
ing for 5 min. After cooling, the reaction products
were analyzed by TLC. As shown in Fig. 4, the trans-
galactosylation product was found to be produced by
transferring a galactosyl residue from
galactopyranoside to glucose. The reaction mixture
was separated into NP- -galactopyranoside,
p
NP-a
-D
-
p
a
-
D
by GC and GC-MS. The identiˆcation was accom-
19)
galactose, glucose, and the transgalactosylation
product by TLC. The product was found in the same
position as the authentic disaccharide, melibiose.
plished using the data of Bjo
ä
rndal et al
.
and
Stellner et al
ed the presence of 1-substituted galactose (1,5-di-(
.
20) The methylation analysis demonstrat-
o
-

1490
T. SRIPUAN et al.
applications because a high temperature and moder-
ate pH are required for processing. Although it is
di‹cult to obtain large amounts of the enzyme from
G. lucidum, we are attempting to clone the gene
encoding this enzyme for large-scale production of
the enzyme.
Many glycosidases are known to have trans-
glycosylation activity, which is considered as a spe-
cial reaction of hydrolysis in which the carbohydrate
moiety of the substrate is transferred to the hydroxyl
group of various compounds instead of water.27)
Transglycosylation activity has been well studied in
many glycosidases. Although the activity is also well
studied in plant28,29) and fungus10,30,31)
a
-galactosi-
dases, the activity is almost unknown in mushroom
-galactosidases. We found that G. lucidum -galac-
tosidase also had transgalactosylation activity. When
-nitrophenyl- -galactopyranoside and glucose
were incubated with this enzyme, melibiose (galac-
tosyl -1,6 glucose) was mainly produced. We are
interested in the fact that ganodran C contained a
galactosyl -1,6 linkage although G. lucidum had
strong -galactosidase activity. The transgalactosyla-
a
a
Fig. 5. Sugar Composition and Linkage Analyses of the Trans-
galactosylation Product.
p
a
-D
(A) Gas chromatograms of TMS derivatives of the product.
a
(a) 1-
2,3,4,6-tetra-
ly methylated alditol acetate derivatives of the product. (a) 1,5-
di-( -acetyl)-2,3,4-tri-( -methyl) galactitol; (b) 1,5,6-tri-(
acetyl)-2,3,4-tri-( -methyl) glucitol.
O
-methyl-2,3,4,6-tetra-
O-TMS-galactose; (b) 1-O-methyl-
O
-TMS-glucose. (B) Gas chromatogram of partial-
a
o
o
o-
a
o
tion activity of this enzyme might involve the synthe-
sis of ganoderan C though a glycoside donor is
unknown.
acetyl)-2,3,4-tri-(
o
-methyl) galactitol) and 1,6-sub-
-acetyl)-2,3,4-tri-(
The substrate speciˆcity of this enzyme is also of
stituted glucose (1,5,6-tri-(
o
o
-
interest. This enzyme is highly active toward
p
-NP-
-galac-
a-galactoside.
a-
methyl) glucitol) in the transgalactosylation product
(Fig. 5B). Therefore, the product would be galactose
galactoside, and slightly active against
o-NP-a
toside, but not active toward -NP-
m
1
ª
6 glucose disaccharide, and since it was degraded
The results suggest that this enzyme clearly recog-
nizes the conformation of aglycon, or enzymatic
inhibition occurred via hindrance of aglycon. Further
investigations are currently ongoing.
by the
by jack-bean
(galactose
a
-galactosidase of green coŠee beans but not
b-galactosidase, it would be melibiose
ª6 glucose). Moreover, the transgalac-
a
1
tosylation product obtained from galactose as an ac-
ceptor was also analyzed and identiˆed as galactose
Acknowledgment
1
ª
6 galactose (data not shown). From these results,
this enzyme could transfer the NP-galactose to the
p
The authors would like to thank the National
Science and Technology Development Agency,
Thailand, for providing a scholarship to graduate
student Thida Sripuan in Thailand. We also grateful-
ly acknowledge the Japan Society for Promotion of
Science (JSPS), for funds to support part of the
research in Japan and the Institute for Science and
Technology Research and Development, Chiang Mai
University, for a grant to support part of the research
in Thailand.
C-6 position of the sugar acceptor.
Discussion
The fruiting bodies of Ganoderma lucidum had
relatively high
a-galactosidase activity and the
enzyme was thermally stable. We were interested in
such enzymatic properties. It is unusual for a
mushroom to have
a-galactosidase activity that is
stronger than other glycosidase activities. Moreover,
the activity had a maximum at pH 6.0 and this value
is higher than the optimum pHs of most mushroom
References
and fungal
distinctive characteristic of the G. lucidum
tosidase was its extreme thermostability. The opti-
mum temperature of this -galactosidase is 70 C and
the enzyme was fully stable after heating at 70 C for
30 min. Even when it was heated at 80 C for 30 min,
almost 30 of its original activity remained. These
properties are advantageous for possible industrial
a
-galactosidases (pH 2.5–5.5).21–26) The
1) Mizuho, T., Suzuki, E., Maki, K., and Tamaki, H.,
Fractionation, chemical modiˆcation, and antitumor
activity of water-soluble polysaccharides of the
a
-galac-
fruiting-body of Ganoderma lucidum. Nippon Noš gei-
a
9
9
kagaku Kaishi (in Japanese), 59, 1143–1152 (1985).
2) Bao, X. F., Wang, X. S., Dong, Q., Fang, J. N., and
Li, X. Y., Structural features of immunologically
active polysaccharides from Ganoderma lucidum.
Phytochemistry, 59, 175–181 (2002).
9
z

Thermostable
a
-Galactosidase from Ganoderma lucidum
1491
3) Tomoda, M., Gonda, R., Kasahara, Y., and Hikino,
H., Glycan structures of ganoderan and C,
18) Pearson, W. A., and Lipman, D. J., Improved tools
for biological sequence comparison. Proc. Natl.
Acad. Sci. USA, 85, 2444–2448 (1988).
B
hypoglycemic glycans of Ganoderma lucidum fruit
bodies. Phytochemistry, 25, 2817–2820 (1986).
4) Furusawa, E., Chou, S. C., Furusawa, S., Hirazumi,
A., and Dong, Y., Antitumor activity of Ganoderma
lucidum, an edible mushroom, on intraperitoneally
implanted Lewis lung carcinoma in synergenic mice.
Phytotherapy Research, 6, 300–304 (1992).
5) Miyazaki, T., and Nishijima, M., Structural exami-
nation of a water soluble, antitumor polysaccharide
of Ganoderma lucidum. Chem. Pharm. Bull., 29,
3611–3616 (1981).
19) Bjoä rndal, H., Hellerqvist, C. G., Lindberg, B., and
Svensson, S., Gas-liquid chromatography and mass
spectrometry in methylation analysis of polysaccha-
rides. Angew. Chem., 82, 643–652 (1970).
20) Stellner, K., Saito, H., and Hakomori, S., Determi-
nation of aminosugar linkages in glycolipids by
methylation-aminosugar linkages of ceramide pen-
tasaccharides of rabbit erythrocytes and of Forssman
antigen. Arch. Biochem. Biophys., 155, 171–178
(1973).
6) Hikino, H., Konno, C., Mirin, Y., and Hayashi, T.,
Isolation and hypoglycemic activity of Ganoderan A
and B, glycan of Ganoderma lucidum fruit bodies.
Planta Medica, 51, 339–340 (1985).
21) Li, Y.-T., and Shetlar, M. R., Occurrence of
a-galac-
tosidase in higher fungi: isolation of -galactosidase
a
from Calvatia cyathiformis. Arch. Biochem.
Biophys., 108, 523–530 (1964).
7) Bao, X. F., Liu, C. P., Fang, J. N., and Li, X. Y.,
Structural and immunological studies of a major
polysaccharide from spores of Ganoderma lucidum.
Carbohydr. Res., 332, 67–74 (2001).
22) Ohtakara, A., Mitsutomi, M., and Uchida, Y.,
Puriˆcation and enzymatic properties of a-galactosi-
dase from Pycnoporus cinnabarinus. Agric. Biol.
Chem., 48, 1319–1327 (1984).
8) Dey, P. M., and Pridham, J. B., Biochemistry of
a
-
D
-
23) Bahl, O. P., and Agrawal, K. M. L., Glycosidases of
Aspergillus niger. Puriˆcation and characterization
galactosidase. In ``Advances in Enzymology'' vol. 36,
ed. Meister, A., Academic Press Inc., New York, pp.
91–130 (1972).
of
a-
and
b-galactosidases and
b-N-acetyl-
glucosaminidase. J. Biol. Chem., 244, 2970–2978
(1969).
9) McCleary, B. V., and Matheson, N. K., a-D-galac-
tosidase activity and galactomannan and galactosyl-
sucrose oligosaccharide depletion in germinating
legume seeds. Phytochemistry, 13, 1747–1757 (1974).
10) Eneyskaya, E. V., Golubev, A. M., Kachurin, A. M.,
Savel'ev, A. N., and Neustroev, K. N., Transglycosy-
24) Shibuya, H., Kobayashi, H., Park, G. G., Komatsu,
Y., Sato, T., Kaneko, R., Nagasaki, H., Yoshida, S.,
Kasamo, K., and Kusakabe, I., Puriˆcation and some
properties of
a-galactosidase from Penicillium pur-
purogenum. Biosci. Biotechnol. Biochem.
,
59,
lation activity of
a
-
D
-galactosidase from Trichoderma
2333–2335 (1995).
reesei: An investigation of the active site. Carbohydr.
Res., 305, 83–91 (1998).
25) Scigelova, M., and Crout, D. H. G., Puriˆcation of
a-galactosidase from Aspergillus niger for application
11) Spangenberg, P., Andre', C., Dion, M., Rabiller, C.,
in the synthesis of complex oligosaccharides. J. Mol.
Catal. Part B: Enzym., 8, 175–181 (2000).
and Mattes, S., Comparative study of new
a-galac-
tosidases in transglycosylation reactions. Carbohydr.
Res., 329, 65–73 (2000).
26) Puchart, V., Vrsanska, M., Bhat, M. K., and Biely,
P., Puriˆcation and characterization of a-galactosi-
12) Koizumi, K., Tanimoto, T., Kubota, Y., and
Kitahata, S., Enzymatic synthesis, isolation, and
dase from a thermophilic fungus, Thermomyces
lanuginosus. Biochim. Biophys. Acta, 1524, 27–37
(2000).
analysis of novel a- and b-galactosyl-cycloisomalto-
octaoses. Carbohydr. Res., 305, 393–400 (1998).
27) Edelman, J., The formation of oligosaccharides by
13) Hashimoto, H., Katayama, C., Goto, M., Okinaga,
enzymic transglycosylation. Adv. Enzymol., 17,
T., and Kitahata, S., Enzymatic synthesis of
a
-linked
189–232 (1956).
galactooligosaccharides using the reverse reaction of
a cell-bound -galactosidase from Candida guillier-
28) Dey, P. M., Transgalactosylation activity of sweet
a
almond a-galactosidase: Synthesis of saccharides.
mondii H-404. Biosci. Biotechnol. Biochem., 59,
179–183 (1995).
Phytochemistry, 18, 35–38 (1979).
29) Koizumi, K., Tanimoto, T., Okada, Y., Hara, K.,
Fujita, K., Hashimoto, H., and Kitahata, S.,
Isolation and characterization of novel heterogeneous
branched cyclomalto-oligosaccharides (cyclodextrins)
14) Bradford, M. M., A rapid and sensitive method for
the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal.
Biochem., 72, 248–254 (1976).
15) Laemmli, U. K., Cleavage of structural proteins dur-
ing the assembly of the head of bacteriophage T4.
Nature, 227, 680–685 (1970).
16) Manabe, T., Tachi, K., Kojima, K., and Okuyama,
T., Two-dimentional electrophoresis of plasma pro-
teins without denaturing agents. J. Biochem., 85,
649–659 (1979).
17) Ciucanu, I., and Kerek, F., A simple and rapid
method for the permethylation of carbohydrates.
Carbohydr. Res., 131, 209–217 (1984).
produced by transgalactosylation with
a-galactosi-
dase from coŠee bean. Carbohydr. Res., 278,
129–142 (1995).
30) Scigelova, M., Singh, S., and Crout, D. H. G.,
Glycosidases-a great synthetic tool. J. Mol. Catal.
Part B: Enzym., 6, 483–494 (1999).
31) Eneyskaya, E. V., Golubev, A. M., Kachurin, A. M.,
Savel'ev, A. N., and Neustroev, K. N., Transglycosy-
lation activity of a- -galactosidase from Trichoderma
D
reesei An investigation of the active site. Carbohydr.
Res., 305, 83–91 (1997).