Purification of an isoflavonoid 7-O-β-apiosyl-glucoside β-glycosidase and its substrates from Dalbergia nigrescens Kurz

Article abstract of DOI:10.1016/j.phytochem.2005.06.024

A β-glycosidase was purified from the seeds of Dalbergia nigescens Kurz based on its ability to hydrolyse p-nitrophenyl β-glucoside and β-fucoside. This enzyme did not hydrolyze various glycosidic substrates efficiently, so it was used to identify its own

Full text of DOI:10.1016/j.phytochem.2005.06.024

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 1880–1889
www.elsevier.com/locate/phytochem
Purification of an isoflavonoid 7-O-b-apiosyl-glucoside
b-glycosidase and its substrates from Dalbergia nigrescens Kurz
a
b
c
a
Phimonphan Chuankhayan , Yanling Hua , Jisnuson Svasti , Santi Sakdarat ,
d
a,
*
Patrick A. Sullivan , James R. Ketudat Cairns
a
Institute of Science, Suranaree University of Technology, Schools of Biochemistry and Chemistry,
111 University Avenue, Nakhon Ratchasima 30000, Thailand
Center for Scientific and Technological Equipment, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
b
c
Center for Excellence in Protein Structure and Function, Department of Biochemistry, Faculty of Science,
Mahidol University, Bangkok 10400, Thailand
d
Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand
Received 11 February 2005; received in revised form 18 May 2005
Available online 10 August 2005
Abstract
A b-glycosidase was purified from the seeds of Dalbergia nigescens Kurz based on its ability to hydrolyse p-nitrophenyl b-gluco-
side and b-fucoside. This enzyme did not hydrolyze various glycosidic substrates efficiently, so it was used to identify its own natural
substrates. Two substrates were identified, isolated and their structures determined as: compound 1, dalpatein 7-O-b-D-apiofurano-
syl-(1 ! 6)-b-D-glucopyranoside and compound 2, 6,20,40,50-tetramethoxy-7-hydroxy-7-O-b-D-apiofuranosyl-(1 ! 6)-b-D-glucopyr-
anoside (dalnigrein7-O-b-D-apiofuranosyl-(1 ! 6)-b-D-glucopyranoside). The b-glycosidase removes the sugar from these glycosides
as a disaccharide, despite its initial identification as a b-glucosidase and b-fucosidase.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Dalbergia nigrescens Kurz; Leguminosae; b-Glucosidase; b-Glycosidase substrate; Isoflavonoid glycoside; Apiosyl glucoside
1. Introduction
plant b-O-linked glycosides are potential b-glucosidase
substrates.
b-glucosidases (3.2.1.21) hydrolyze glycosidic bonds
between non-reducing terminal glucose residues and
alcohols of various structures. The aglycones released
by plant b-glucosidases that have been identified include
saccharides, hydroxynitriles, hydroxamic acids, naph-
thoquinones, alkaloids, and isoflavonoids, among others
(Esen, 1993; Poulton, 1990; Babcock and Esen, 1994;
Duroux et al., 1998; Warzecha et al., 2000; Svasti
et al., 1999). Since some plant b-glucosidases are able
to catalyze the release of sugars other than glucose, all
Many isoflavonoid glycosides have been isolated
from tropical trees of the genus Dalbergia, family legu-
minosae. These include rotenoid derivatives (Chibber
and Khera, 1979; Svasti et al., 1999), and other isoflav-
one derivatives (Mathias et al., 1998; Rajasekhara Rao
and Srinivasa Rao, 1991; Sharma et al., 1980; Farag
et al., 1999). These compounds include both O-linked
and C-linked glycosides, simple glucosides and oligosac-
charide glycosides.
Previously, Srisomsap et al. (1996) described the puri-
fication and characterization of a b-glucosidase from
Dalbergia cochinchinesis Pierre seeds and Svasti et al.
(1999) described the use of this b-glucosidase to identify
its substrate, dalcochinin-12-O-b-D-glucoside from the
*
Corresponding author. Tel: +66 44 224304; fax: +66 224185.
E-mail address: cairns@ccs.sut.ac.th (J.R. Ketudat Cairns).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.06.024

P. Chuankhayan et al. / Phytochemistry 66 (2005) 1880–1889
1881
same seeds. Here, we have used the same approach to
identify a b-glycosidase with distinct specificity and its
substrates and products from the seed of Dalbergia
nigrescens Kurz.
The purified enzyme gave a single band with apparent
molecular weight of 62–63 kDa on SDS–PAGE (Fig. 1).
Gel filtration of the native enzyme on Sephacryl S-300
gave a molecular weight of approximately 240 kDa, so
the native enzyme appears to consist of 4 subunits.
Non-denaturing polyacrylamide gel electrophoresis of
the purified enzyme followed by activity staining with
4-methylumbelliferyl (4-MU) substrates, 4-MU-b-D-glu-
coside, 4-MU-b-D-fucoside and 4-MU-b-D-galactoside,
showed a single fluorogenic product band for each sub-
strate at the same position as the main protein band
(Fig. 2). This data suggests that the purified enzyme con-
sists of a single component, which cleaves b-glucosides,
b-fucosides, and b-galactosides.
2. Results and discussion
In preliminary experiments, b-glucosidase from D.
cochinchinesis was shown to hydrolyze a glycoside from
D. nigrescens seed extract, but not as well as crude D.
nigrescens b-glucosidase, as judged by the shift in mobil-
ity of a spot on TLC. Therefore, the D. nigrescens b-glu-
cosidase was purified and used to identify its substrates.
Sequencing of the N-terminus of the purified protein
gave the sequence ATITEV in rather low yield. The last
5 residues of this sequence matches residues 10–14 of D.
cochinchinensis b-glucosidase (Ketudat Cairns et al.,
2.1. Purification of b-glucosidase/b-fucosidase from
D. nigrescens Kurz seeds
The enzyme was extracted from seeds and purified by
35–75% ammonium sulfate fractionation, DEAE anion
exchange chromatography and S-300 gel filtration chro-
matography, as described in the methods. Both p-nitro-
phenol (pNP)-b-D-glucoside and pNP-b-D-fucoside
hydrolysis activities were determined during the purifi-
cation. The yields for b-glucosidase and b-fucosidase
were similar, but the total and specific activities of b-
fucosidase were twice as much as those of b-glucosidase
at each step (Table 1). The enzyme was purified 33 and
49 fold over the crude extract for b-glucosidase and b-
fucosidase, respectively, with 2–3% yield of both total
activities, or about 2 mg of enzyme per 50 g of seed.
Large amounts of black polyphenolic compounds were
found in the seeds, which required extraction in reducing
agents and were not completely eliminated until after the
DEAE chromatography. Removal of these polyphenolic
compounds may partially account for the relatively low
protein yields obtained.
Fig. 1. SDS–PAGE analysis of purified b-glucosidase/b-fucosidase
enzyme from D. nigrescens seeds. Lane M, Low-range protein markers;
1, crude extract; 2, 35–75% (NH4)2SO4 fractionated; 3, DEAE pooled
fractions; 4, S 300 gel filtration fraction.
Table 1
Purification of b-glucosidase/b-fucosidase enzyme from Dalbergia nigrescens Kurz
Fraction
Total activity (nkat)
Total protein (mg)
3040
Specific activity (nkat/mg)
Purification (fold)
Yield (%)
Crude extract
b-Glucosidase
b-Fucosidase
6130
9470
2.02
3.11
1.00
1.00
100
100
35–75% (NH4)2SO4
b-Glucosidase
b-Fucosidase
1390
12.3
1.79
4220
8680
3.04
6.26
1.50
2.01
68.9
91.6
DEAE
b-Glucosidase
b-Fucosidase
574
1010
46.8
82.2
23.1
26.4
9.37
10.6
S300 gel filtration
b-Glucosidase
b-Fucosidase
119
271
66.6
152
33.0
48.9
1.94
2.86
Fifty grams of seeds were used in purification. Enzyme reactions were performed with 1 mM pNPꢀb-D-glucoside or 1 mM pNPꢀb-D-fucoside in
0.1 M sodium acetate, pH 5.0 at 30 ꢁC for 10 min.

1882
P. Chuankhayan et al. / Phytochemistry 66 (2005) 1880–1889
Fig. 2. Activity staining of non-denaturing polyacrylamide gel electrophoresis of D. nigrescens b-glucosidase/b-fucosidase enzyme: (a) activity
staining with 1 mM of 4-MU-b-D-glucoside; lane 1, 4.8 lg; lane 2, 2.4 lg; lane 3, 1.2 lg of enzyme; (b) protein stained with Coomassie blue R-250;
lane 1, 4.8 lg; lane 2, 2.4 lg; lane 3, 1.2 lg of enzyme. C. 10 lg of enzyme stained with 1 mM 4-MU-b-D-fucoside (1) and 1 mM 4-MU-b-D-
galactoside (2).
2000). Four tryptic peptides were also sequenced, and
they also had high similarity to the sequence of D.
cochinchinensis b-glucosidase, as shown in Table 2.
The sixth residue of the Tryp 2 peptide gave only a
low yield of the Ile from the preceding step, which, to-
gether with its location 2 residues before Thr and the
fact the corresponding residue in D. cochinchinensis is
Asn, suggests it may be an N-linked glycosylation site.
In total, the D. nigrescens b-glycosidase was identical
with D. cochinchinensis b-glucosidase at 51 of 60 resi-
dues sequenced, which indicates this enzyme is also a
family 1 glycosyl hydrolase (Henrissat, 1991).
was 65 ꢁC. To obtain a preliminary assessment of sugar
specificity, pNP-glycosides were tested as substrates at
5 mM (Table 3). The enzyme showed the highest activity
toward pNP-b-D-fucoside and pNP-b-D-glucoside,
which is similar to D. cochinchinensis b-glucosidase
(Srisomsap et al., 1996). Other pNP-glycosides were
hydrolyzed at less than 10% of the rate of pNP-b-D-glu-
coside. The enzyme could not significantly hydrolyze
pNP-b-cellobioside.
Release of glucose from available natural glycosides
and oligosaccharides was also tested. The rates of
hydrolysis were extremely low with laminaribiose hydro-
lyzed at 1.0%, linamarin at 0.37% and cellobiose at
0.33% the rate of pNP-b-D-glucoside hydrolysis. No
hydrolysis of laminaritriose, cellotriose, salicin, dhurrin,
DIMBOA glucoside, sophorose, torvoside A and amyg-
dalin could be observed. The enzyme did cleave dalcoch-
inin-80-O-b-D-glucoside, the natural substrate of D.
cochinchinensis b-glucosidase, as judged by TLC analysis
(data not shown). However, the activity of the D. nigres-
cens enzyme toward this substrate was rather low com-
pared to that of D. cochinchinensis b-glucosidase.
The effect of various compounds was tested on the
hydrolysis of pNP-b-D-glucoside and pNP-b-D-fucoside
by the purified D. nigrescens glycosidase. Neither the
metals tested, FeSO4, CaCl2, HgCl2, MnCl2, and
2.2. Catalytic activity of purified b-glucosidase/b-
jucosidase
The enzyme was found to have a pH optimum of
5.0–6.0 with ca half maximal activity at pH 3.5 and
6.5. In the standard 10 min. assay with 1 mM pNP-
glucoside and pNP-fucoside, the temperature optimum
Table 2
Peptide sequences from D. nigrescens b-glycosidase
Peptide
Sequence
N-terminus
D. nigrescens
ATITEV
ETITEV
D. cochinchinensis 9–14
Table 3
Tryp 1
D. nigrescens
Comparison of hydrolysis of synthetic substrates by D. nigrescens and
D. cochinchinensis b-glucosidases
YMNLDAYR
DMNLDAYR
D. cochinchinensis 82–90
Substrate
D. nigrescens %
Relative activity
D. cochinchinensis %
Relative activity
Tryp 2
D. nigrescens
ASGGIISTGVD
D. cochinchinensis 104–114 VSGGINQTGVD
pNPꢀb-D-glucoside
pNPꢀb-D-fucoside
100
124
3.97
7.55
1.91
0.68
0.47
0.40
100
124
8.95
3.91
4.89
0.02
Not determined
0.26
Not determined
Tryp 3
D. nigrescens
pNPꢀb-D-galactoside
pNPꢀb-D-xyloside
LINETLANGI
D. cochinchinensis 119–128 LINESLANGI
pNPꢀa-L-arabinoside
pNPꢀb-D-thioglucoside
pNPꢀb-L-arabinoside
pNPꢀb-D-mannoside
pNPꢀb-cellobioside
Enzyme reactions were performed with 5 mM pNP-glycosides, as
described in the methods. Relative activities for D. cochinchinensis b-
glucosidase are taken from Srisomsap et al. (1996).
Tryp 4
D. nigrescens
HWITVNEPSIFTMNGYAYGIFAPGR
D. cochinchinensis 176–200 HWITLNEPSIFTANGYAYGMFAPGR
Not detectable
The N-terminus and tryptic peptides of the D. nigrescens b-glycosidase
are shown above the corresponding sequence from the D. cochinchin-
ensis b-glucosidase numbered according to its position in the mature
protein.

P. Chuankhayan et al. / Phytochemistry 66 (2005) 1880–1889
1883
ZnSO4, nor EDTA had a significant effect on either b-
glucosidase or b-fucosidase activities at 1 mM
concentration. In contrast, HgCl2 had a strong inhib-
itory effect on D. cochinchinensis b-glucosidase and
garbonzo bean isoflavonoid b-glucosidases (Srisomsap
et al., 1996, Ho¨sel and Barz, 1975). Inhibition by
1 mM d-gluconolactone was also weaker than its effect
on D. cochinchinensis b-glucosidase, with only 34%
inhibition of b-glucosidase activity.
and 315 nm). From FTMS, this compound showed a
molecular related ion peak at m/z 659 [M + Na]+, which
corresponded to the molecular formula C29O16H32 and
to the 29 peaks in the 13C NMR spectrum. Future frag-
mentation of this signal gave an ion at m/z 365.
The formula C18H13O7 of the aglycone is consistent
with an isoflavone (C6–C3–C6) which contains two
methoxy groups and one O–CH2–O at C-70. The 294
amu derived from mass subtraction indicated 2 sugar
rings of five and six carbon, which corresponded to
the 11 carbon signals obtained by subtraction of the
aglycone carbon signals from the total number of gly-
coside 13C signals observed in the 13C NMR (Table
4). After hydrolysis with enzyme or with acid, the
spectrum of purified aglycone, compound 1a, lacked
these signals. The HMBC spectrum of compound 1 re-
vealed cross peaks between two methoxy methyl pro-
tons at d 3.66 and d 3.93 with the C-6 aromatic
carbon position at d 147.31 and C-20 position at d
152.77. The 1H NMR spectrum showed five singlet
signals at d 8.19,d 7.44,d 7.32,d 6.87, and d 6.83,
which are attributable to hydrogens at carbons C-2,
C-5, C-8, C-30 and C-60 of an isoflavone derivative,
as showed in Table 1. The COSY experiment showed
no proton cross peak, which confirmed the para posi-
2.3. Identification of natural substrates and products
Hydrolysis of methanolic extracts of D. nigrescens
seeds with the purified enzyme resulted in a shift in a
few fluorescent and UV-absorbing spots on analytical
TLC. Fractionation of the extract by LH-20 gel filtration
chromatography, followed by preparative TLC allowed
the two major substrates to be purified and character-
ized. Another lower abundance substrate, which was
seen as a blue fluorescent spot just ahead of the com-
pound 1 substrate on TLC, coeluted with compound 1
on LH-20. This compound also coeluted with compound
1 in reverse-phase HPLC. In order to purify compound
1, this substrate was digested with D. cochinchinensis b-
glucosidase, which rapidly digested it, but not compound
1. This digestion allowed the purification of compound 1,
but not the minor substrate or its aglycone, due to their
low abundance. It is possible that other low abundance
glycoside substrates were present, but compounds 1
and 2 appeared to be the most abundant substrates.
1
tion of the protons at C-8 and C-5. The H NMR sig-
nals for the aglycone in DMSO-d6 matched those
previously reported for dalpatein from Dalbergia pan-
iculata (Adinarayana and Rajasekhara Rao, 1975; Ta-
hara et al., 1993; Radhakrishniah, 1973).
A b-D-apiofuranoside moiety was determined by
chemical shift at C-1000 (d 109.48), which is ascribable
to the anomeric dioxymethine carbon on an apiose
ring, along with the carbonic signals of one quater-
nary carbon at C-3000 (d 78.60), two oxymethylenes at
C-4000 (d 73.30) and C-5000 (d 63.32), and one oxyme-
thine at C-2000 (d 72.98). The remaining 13C signals
which corresponded to carbonic carbon were used to
elucidate a b-D-glucopyranoside moiety with one ano-
meric dioxymethine at C-1000 (d 99.90), four mono-
oxymethine at C-2000 (d 72.98), C-3000 (d 76.67), C-4000
(d 69.80) and C-5000 (d 75.65) carbons. The chemical
shift at C-6000 (d 67.85) was used to define the disac-
charide linkage as apiofuranosyl (C-1000 ! C-6000) be-
5'''
HOCH2
H
O
6''
CH2
O
1'''
H
4'''
H
H
5''
H
H
8
O
H
2'''
3'''
H
1'
O
O
2
1''
H
9
H
OH
OMe
2'
4''
HO
OH OH
7
3
H
O
2''
3''
H
10
6
MeO
4
3'
4'
5
OH
6'
O
O
H
compound 1
compound 1a
H
2
5'
O
7'
H
8
H
HO
9
OMe
2'
cause the chemical shift of
a
monosaccharide
7
3
oxymethylene group appears at about dC62 (Mathias
et al., 1998). Also, the chemical shift at C-1000 (d
109.48) indicated C-1 anomeric orientation of apiose
as b-configuration because the 13C NMR of a-D-api-
ose would appear at about dC104.5 (Takahashi
et al., 2001). The NMR spectral data of the sugar part
are in agreement with those of tectorigenin 7-O-[b-D-
apiofuranosyl-(1 ! 6)-b-D-glucopyranoside] from Dal-
bergia sissoo (Farag et al., 1999).
H
1'
10
6
MeO
4
3'
4'
5
6'
O
H
H
O
5'
O
7'
Compound 1
Compound 1 glycoside was obtained as a pale yellow
powder. The UV spectrum showed the characteristic
The aglycone of compound 1 (compound la), 6,20-
dimethoxy-40,50-methylenedioxyisoflavone, was the same
MeOH
max
absorption of an isoflavone compound (k
260

1884
P. Chuankhayan et al. / Phytochemistry 66 (2005) 1880–1889
Table 4
1H and 13C NMR spectral data of compounds 1, 2 and 2a
Position
Compound 1 (DMSO-d6)
Compound 2 (D2O)
Compound 2a (CD3OD)
13C
1H
HMBC
13C
1H
HMBC
13C
1H
HMBC
2
3
4
5
6
7
8
9
154.42 CH
121.03 C
174.66 C
104.63 CH
147.31 C
151.41 C
103.72 CH
151.19 C
117.68 C
112.92 C
152.77 C
95.4 CH
147.79 C
140.26 C
110.90 CH
101.08 CH
99.90 CH
72.98 CH
76.67 CH
69.80 CH
75.65 CH
67.85 CH2
109.48 CH
75.85 CH
78.60 C
8.19(s)
156.40 CH
121.05 C
177.47 C
105.21 CH
151.43 C
152.29 C
104.10 CH
147.58 C
111.92 C
115.60 C
152.16 C
98.72 CH
149.75 C
142.35 C
118.11 CH
8.03(s)
156.24 CH
120.52 C
176.48 C
102.72 CH
152.92 C
153.30 C
101.97 CH
150.18C
111.02 C
113.95 C
153.30 C
98.76 CH
152.06 C
143.10 CH
116.90 CH
7.87(s)
2,30,60
2,5,8
8
5,8,OMe
5,8
2,30,60
2,5,8
2,30,60
2,5,8
7.44(s)
7.32(s)
7.17(s)
7.35(s)
8
6.50(s)
7.33(s)
8
5,8,OMe
5,8
5
2,5,8
2,5,8
2,30,60
30,60,OMe
60
30.60,OMe
30,60,OMe
30
5,8,OMe
5,8
5
2,5,8
2,5,8
2,30,60
30,60,OMe
60
30,60,OMe
30,60,OMe
30
5
2,5,8
2,5,8
2,30,60
30,60,OMe
60
30,60,70
30,60,70
30
10
10
20
30
6.87(s)
6.71(s)
6.86(s)
6.73(s)
693(s)
40
50
600
6.83(s)
6.0(s)
5.08(d)J = 6.84
3.33
3.34
3.15
3.60
3.88(d)J = 9.19
4.80(d)J = 2.94
3.77
700
100
100.26 CH
72.94 CH
77.09 CH
69.57 CH
75.41 CH
67.76 CH2
109.38 CH
75.69 CH
5.21(d) J = 7.00
3.65
3.86
3.59
200
300
4
500
3.76
600
4.02(d)J = 9.15
5.02(d)J = 2.87
3.69
1000
2000
3000
79.51 C
4000
73.30 CH2
63.32 CH2
55.75
3.93
3.37
3.93
3.66
73.87 CH2
64.02 CH2
6-OMe 56.67
20-OMe 56.85
40-OMe 56.17
50-OMe 56.38
3.92
3.53
3.73
3.68
3.85
3.82
5000
6-OMe
20-OMe
6-OMe 55.98
20-OMe 56.35
40-OMe 54.68
50-OMe 55.65
3.80
3.75
3.95
3.90
56.63
as dalpatein which has been isolated from D. paniculata
and Piscidia erythrina. But compound 1 contained a
disaccharide moiety, where as previously only the gluco-
side (dalpatin) has been identified (Adinaraya and Raj-
asekhara Rao, 1972).
Compound 2 glycoside was also obtained as a pale
yellow powder and had a molecular related ion peak
[M + Na]+ at m/z 675 in MS, which corresponded to
the molecular formula C30O16H36. The future fragmenta-
tion of this signal gave an ion at m/z 381. The UV spec-
trum showed the characteristic absorption of an
MeOH
max
1
5'''
isoflavone compound (k
260 and 315 nm). The H
HOCH2
NMR spectrum revealed 5 singlet signals at d 7.93,d
7.39,d 6.58,d 6.80 and d 6.96 attributable to hydrogen
at carbons C-2, C-5, C-8, C-30 and C-60 of an isoflavone
derivative. The gHMBC spectrum had cross peaks be-
tween 4 methoxy proton groups which had signals at
C-20 (d 3.68(s)), C-6 (d 3.71(s)), C-40(d 3.85(s)) and C-
50(d 3.82) and carbon at C-20 (d 152.16), C-6 (d 151.43),
C-40 (d 149.75) and C-50 (d 142.35). The 11 carbon signals
in the region of sugar corresponded to one pentose and
one hexose sugar ring, with nearly the same spectrum
as in compound 1, though the compound 2 structure
was solved in D2O rather than DMSO-d6 as solvent.
From this comparison, the two compounds appeared
to have the same sugar moiety structure. Again, these
sugars were removed by hydrolysis with D. nigrescens
b-glucosidase to give compound 2a. NMR of compound
2a was done in CD3OD because it was less soluble in
H
O
6''
CH2
O
1'''
H
4'''
H
H
5''
H
8
H
O
H
2'''
3'''
2
H
1'
O
O
9
1''
H
H
OH
OMe
2'
4''
HO
OH OH
7
3
H
2''
3''
H
10
6
MeO
4
3'
5
OH
6'
O
O
H
compound 2
4'
H
2
OMe
5'
OMe
H
8
H
HO
9
OMe
2'
7
3
compound 2 a
H
1'
10
6
MeO
4
3'
5
6'
O
H
4'
OMe
H
5'
OMe
Compound 2

P. Chuankhayan et al. / Phytochemistry 66 (2005) 1880–1889
1885
D2O than compound 2. The 1H NMR spectrum showed
5 singlet signals at d 7.87,d 6.50,d 7.33,d 6.73 and d 6.93
indicating the hydrogen atoms at carbons C-2 (d 156.24),
C-5 (d 102.72), C-8 (d 101.97), C-3 (d 98.76) and C-60(d
116.90). The gHMBC spectrum had cross peaks between
4 methoxy proton groups which had signals at C-20 d
3.75(s), C-40d 3.95(s), C-50 d 3.90(s) and C-60 (d 3.80)
and carbons at C-20 (d 153.30), C-40(d 152.06), C-50 (d
143.10) and C-6 (d 152.92). The DEPT experiment indi-
cated 7 methyl carbon peaks and 3 methylene carbon
peaks, which were in the sugarÕs chemical shifts region
of compound 2.
respectively. In addition, the enzyme showed much
higher kcat values for compounds 1 and 2 than for
pNP-b-D-glucoside and pNP-b-D-fucoside. In contrast,
D. cochinchinensis b-glucosidase, cassava linamarase
and almond b-glucosidase could not significantly hydro-
lyze these substrates (data not shown). The high specific-
ity of D. nigresecens b-glycosidase for compounds 1 and
2 and the inability of related enzymes to hydrolyze them
indicate that they are likely to be the natural substrates of
this enzyme.
When the products of the hydrolysis of compounds 1
and 2 were characterized by TLC, only one sugar spot
was seen. This sugar migrated in a position similar to
standard disaccharides when run on TLC or when run
on GC after trimethylsilylation. When the sugar was
purified and hydrolyzed with acid, apiose and glucose
were obtained. This indicates the enzyme removed the
disaccharide from the glycoside, in contrast to the expec-
tation that it would cut off one sugar at a time, as do most
b-glucosidases. Ho¨sel and Barz (1975) previously ob-
served cleavage of biochanin-glucoapioside to biochanin
and disaccharide by garbonzo bean isoflavonoid b-glu-
cosidases. It is not so surprising that a family 1 enzyme
should be a disaccharidase, since the primeverosidase
(EC 3.2.1.149) from tea also belongs to this family
(Mizutani et al., 2002). However, since the active site of
these enzymes is a funnel-shaped pocket with the sugar
binding site on the inside (Barrett et al., 1995), such
disaccharidases presumably have a larger pocket for
the sugar than monosaccharide-specific enzymes in this
family.
The aglycone structure of compound 2a, 7-O-glyco-
syl-7-hydroxy-20,40,50,6-tetramethoxyisoflavone
was
similar to that of dalpaniculin-C-glycosylisoflavone
from D. paniculata seeds, which is 8-C-b-D-glucopyr-
anosyl-5-7-dihydroxy-20,40,50,6-tetramethoxyisoflavone
(Rajasekhara Rao and Srinivasa Rao, 1991). How-
ever, compound 2a (aglycone) is linked with the gly-
cone through the oxygen at C-7 in compound 2,
rather than through a C–C bond at C-8.
In order to verify the sugars, the glycoside was acid
hydrolyzed, trimethylsilylated and submitted to GC.
The gas chromatogram of the trimethylsilyl derivatives
of sugars in the sample showed 4 peaks with the same rel-
ative retention time as the apiose standard and 2 with the
same retention times as those in the glucose standard.
The mass fragmentation patterns of these peaks also
matched those of the standards and those for glucose
matched the a- and b-D-glucopyranose forms in the
GC-MS library.
In the case of primeverosidase, the enzyme is spe-
cific for the disaccharide (Mizutani et al., 2002), while
garbonzo bean isoflavonoid b-glucosidase hydrolyzed
isoflavonoid glucosides better than apiosoglucosides
(Ho¨sel and Barz, 1975). To see if D. nigrescens isofl-
avonoid b-glycosidase preferred glucosides, two isofl-
avonoid glucosides, daidzin and genistin, were tested.
The two were hydrolyzed with Km values somewhat
lower and kcat values somewhat higher than the natu-
ral substrates (Table 5), indicating D. nigrescens b-gly-
cosidase may prefer glucosides, as well. It is possible
such glucoside substrates are also present in the plant,
though they were not identified in this study. TLC
analysis indicated the enzyme could also hydrolyze
600-O-malonyl-genticin, though less efficiently than the
isoflavonoid glucosides (data not shown).
2.4. Kinetics and mechanism of hydrolysis of natural
substrates
The Km and kcat values were determined by using
pNP-b-D-glucoside, pNP-b-D-fucoside and the 2 purified
natural substrates from D. nigrescens seeds (Table 5).
The kcat values of native enzyme were calculated per sub-
unit assuming a molecular weight of 62 kDa. The best
substrates for the enzyme were the
2
natural
D. nigrescens glycosides, compounds 1 and 2. The Km
values were 0.5 mM for compound 1 and 0.7 mM for
compound 2. These Km values were about 3 and 30 times
lower than pNP-b-D-fucoside and pNP-b-D-glucoside,
Table 5
Kinetic properties of purified D. nigrescens b-glycosidase
Thus, the D. nigrescens b-glycosidase can hydrolyze
isoflavonoid 7-O-b-glucosides, 7-O-b-apiosylglucosides
and 7-O-b-malonylglucosides.
Substrate
Km (mM) kcat (sꢀ1
)
kcat/Km (Mꢀ1 sꢀ1
)
pNP-b-D-glucoside
pNP-b-D-fucoside
Compound 1 glycoside
Compound 2 glycoside
Daidzin
14.7
1.8
0.5
0.7
0.11
0.09
10.4
7.0
465
334
480
500
876
4020
9.9 · 105
4.4 · 105
4.4 · 106
5.6 · 106
3. Conclusion
Genistin
The b-glycosidase purified from D. nigrescens Kurz
The values of kcat and kcat/Km were estimated assuming a subunit
molecular weight of 62 kDa.
was found to be a glycosyl hydrolase family 1 isoflavonoid

1886
P. Chuankhayan et al. / Phytochemistry 66 (2005) 1880–1889
b-apioglucosidase. This is the first time this activity has
been described for this family of enzymes, though it
includes a wide variety of b-glycosidases. The enzyme
also hydrolyzed various pNP-glycosides, although with
lower activity. The enzyme had a subunit molecular
weight of 62–63 kD and appeared to be a tetramer,
based on its native molecular weight. These properties,
as well as the sequences of peptides from the protein,
were similar to D. cochinchinensis b-glucosidase. Two
substrates of D. nigrescens Kurz b-glycosidase were iso-
lated and found to be novel isoflavonoid apiosyl gluco-
sides, from which it removes two sugars at a time,
though it can also remove a single glucose from isoflavo-
noid 7-O-glucosides. This is in contrast to the apparently
similar D. cochinchinensis Pierre b-glucosidase, for which
the substrate is a simple rotenoid glucoside, dalcochinin
120-O-b-D-glucoside (Svasti et al., 1999). Both enzymes
are apparently involved in isoflavonoid metabolism, but
the roles of these enzymes from related species may be
somewhat different.
Sigma Chemical (St. Louis, MO, USA). Daidzin,
daidzein, genistin, genistein and 600-O-malonylgenistin
were purchased from LC Laboratories (Woburn, MA,
USA). All other reagents were analytical grade or
better.
4.4. Enzyme purification
Seeds were surface sterilized with 0.1% hypochlorite
for 10 min, then washed with distilled water and soaked
overnight. After this, all procedures were done at 4 ꢁC.
Seeds were homogenized with 2 volumes of buffer 1
(0.025 M Tris–HCl, pH 8.0, 70% (NH4)2SO4, 10 mM
ascorbic acid, 0.025 M b-mercaptoethanol, 1 mM phen-
ylmethylsulfonyl fluoride (PMSF)). After filtration with
cheese cloth, the solids were extracted 2 more times.
Then, the solids were extracted with 4 volumes of buffer
2 (0.025 M Tris–HCl, pH 8.0, 0.2 M NaCl, 10 mM ascor-
bic acid, 0.025 M b-mercaptoethanol, 1 mM PMSF), and
centrifuged at 12,000 rpm for 15 min at 4 ꢁC. Polyvinyl-
polypyrrolidone (PVPP) was added to the supernatant
up to 2% and stirred at 4 ꢁC for 1 h. The homogenate
was centrifuged at 15,000 rpm for 20 min at 4 ꢁC, and
the pellet was discarded. Activated Dowex 2 · 8 resin
was added to the supernatant to 25% (w/v), mixed by
stirring and removed by centrifugation at 15,000 rpm
for 20 min at 4 ꢁC. The protein was fractionated from
the supernatant by 35–75% (NH4)2SO4 precipitation,
and the resulting protein pellet was resuspended in
25 mM Tris–HCl, pH 7.0 before dialysis against the same
buffer overnight. The (NH4)2SO4 fractionated protein
was separated on a DEAE-Sepharose anion exchange
column (1.25 · 23.5 cm,115 cm3) with a linear gradient
of 0–1 M NaCl in 25 mM Tris–HCl, pH 7.0, at a flow rate
of 0.65 ml/min. Fractions containing b-glucosidase and
b-fucosidase activity were pooled, concentrated by ultra-
filtration (Amicon YM-50, Millipore Corp., Billerica,
MA, USA) and fractionated by chromatography in a
Sephacryl S-300 (Amersham Pharmacia) gel filtration
column (1.5 · 25 cm,150 cm3) equilibrated with 50 mM
Tris–HCl pH 7.0, 0.3 M NaCl at a flow rate 0.8 ml/min.
4. Experimental
4.1. General
The structure of the purified compounds were deter-
mined by 1H NMR, 13C NMR, COSY, gHMBC,
gHMQC and DEPT on a 300 MHz Varian UNITY In-
ova spectrometer (Innova, Darmstadt, Germany). Mass
spectrometry of glycosides and aglycones was done on
an Esquire-LC Mass spectrometer (Bruker Daltonics,
Germany). Thin-layer chromatography and preparative
thin-layer chromatography were performed on silica gel
60 F254 (Merck, Darmstadt, Germany). Column chro-
matography was performed with Sephadex LH-20
(Amersham Biosciences, Uppsala, Sweden). GC–MS
analysis of sugars was done on a Varian-CP-3800 Gas
Chromatograph and MS-1200L Quadrupole Spectrome-
ter (Varian, CA, USA.) with a Varian-FactorFour Cap-
illary Column, VF-5 ms 30 M · 0.25 MM I.D.
4.2. Plant material
4.5. Protein analysis
D. nigrescens seeds were collected from Suranaree
University of Technology, Nakhon Ratchasima, Thai-
land, in January 2003.
Protein concentration was determined by the Lowry
method (1951) with bovine serum albumin as standard,
while column effluents were screened for protein by
measurement of A280. Denaturing SDS–PAGE was
performed by the general method of Laemmli (1970)
on 10% polyacrylamide separating gels with Bio-RAD
Low-range protein markers (Bio-RAD, Corp., Hercules,
CA, USA), and stained with Coomassie Brilliant Blue
R250. Non-denaturing polyacrylamide gel electrophore-
sis, was performed with 7% polyacrylamide separating
gels in Laemmli buffer without SDS. Separate gels were
stained for b-glucosidase, b-fucosidase and b-galactosi-
4.3. Other materials
D(+)-Glucose was purchased from Scharlau (Schar-
lau Chemie S.A., Barcelona, Spain) and apiose from
Omicron (Biochemicals Inc., South Bend, IN, USA).
Commercial substrates, 2-mercaptoethanol, polyvinyl-
polypyrolidine (PVPP), phenyl methyl sulfonyl fluoride
(PMSF), and Dowex 2 · 8 resin were obtained from

P. Chuankhayan et al. / Phytochemistry 66 (2005) 1880–1889
1887
dase activities with 1 mM 4-methylumbelliferyl-b-glyco-
4.8. Compound 1 and 2 extraction and isolation
sides. The fluorogenic product bands were detected with
a Fluor-STM MultiImager (Bio-RAD). The native
molecular weight of the protein was estimated by using
Sephacryl S-300 gel filtration chromatography cali-
brated with b-amylase (200 kDa), alcohol dehydroge-
nase (150 kDa), carbonic anhydrase (29 kDa), and
cytochrome C (12.4 kDa) as standards.
The N-terminus of the purified protein was deter-
mined on an ABI 473A Peptide Sequencer (Applied Bio-
Systems Inc., Foster City, CA, USA). The protein was
digested with trypsin (Promega Corp., Madison, WI,
USA), and the whole protein or peptides were isolated
by HPLC on a Vydac-C4 (4.6 · 250 mm (5 lm)) reverse
phase column (Grace Vydac, Columbia, MD, USA),
dried onto a glass fiber filter and sequenced.
The powder was extracted with 3 volumes of absolute
methanol by stirring overnight at room temperature.
The extract was filtered and partitioned with 1 volume
of hexane. The methanol fraction was dried by rotary
evaporator and the residue dissolved with water. The
water fraction was extracted with 2.5 volumes of ethyl
acetate and then dried by speed vacuum. The dried res-
idue was dissolved with methanol and separated by
chromatography on a 1.2 · 85 cm Sephadex LH-20 gel
filtration column. The compounds were eluted with
methanol collecting 5 mL fractions, and the fractions
containing b-glucosidase substrates were pooled. The
b-glucosidase substrates were identified by incubating
aliquots of fractions (10% by volume) with and without
b-glucosidase in 0.1 M NaOAc (pH 5.0) 10 min at 30 ꢁC.
The products were separated on analytical silica gel
60 F254 aluminum TLC sheets developed twice with sol-
vent A, EtOAc/methanol/H2O/acetic acid 15:2:1:2 (by
vol). The sheets were visualized under 254 nm wave-
length UV light to identify spots with shifted mobility
after enzymatic digest.
Compounds 1 and 2 were purified using the prepara-
tive thin-layer chromatography by developing twice with
solvent A. The LH-20 column fractions containing com-
pound 1 were incubated overnight in 0.1 M NaOAc buf-
fer, pH 5.0, with D. cochinchinensis b-glucosidase to
hydrolyze low abundance glycoside contaminants before
TLC purification of the compound 1 glycoside. The
compounds 1 and 2 glycosides were digested with 0.1
unit of D. nigrescens b-glucosidase to the aglycones by
incubating in 0.1 M sodium acetate buffer pH 5.0 at
30 ꢁC, overnight. The aglycone of compound 2 was puri-
fied by preparative silica gel 60 F254 TLC using solvent
B, EtOAc/methanol/H2O 5:2:1 (by vol).
4.6. Determination of b-glycosidase activity
Enzyme reactions were performed in 0.1 M NaOAc
buffer, pH 5.0 containing p-nitrophenyl-b-D-glycoside.
The reaction was incubated at 30 ꢁC for 10 min in the
standard assay or various time points for kinetic determi-
nations and stopped with 2 M sodium carbonate. The re-
leased p-nitrophenol (pNP) was determined by
measuring absorbance at 400 nm (Montreuil et al.,
1986). The pH optimum was determined over the range
of pH 3–8.5 in 0.5 pH unit steps in 0.1 M Na citrate,
NaOAc, and potassium phosphate buffers at 30 ꢁC.
The temperature optimum was determined with 1 mM
pNP-b-D-glucoside and 1 mM pNP-b-D-fucoside in
0.1 M NaOAc over a range of 35–85 ꢁC in a 10 min as-
say. Hydrolysis of commercially available natural gluco-
sides was measured by glucose release, which was
quantitated by a PGO glucose oxidase assay (Sigma Fine
Chemicals, St Louis, MO, USA). In the case of daidzin
and genistin, the substrates had to be dissolved in
DMSO, which was added to 5% or less to the assays.
At this concentration, it had little effect on enzyme activ-
ity. The PGO assay had to be finished at 10 min to avoid
interference. Enzyme kinetics were analyzed by linear
regression using the method of Lineweaver and Burk
(1934) and by non-linear regression with the Enzfitter
computer program (Elsevier Biosoft, Cambridge, UK).
4.9. HPLC and TLC analysis of hydrolysis products
These natural substrates, S1 and S2 were subjected to
enzyme hydrolysis in the standard assay described
above. The reaction products were separated and quan-
tified with an Eclipse XDB-C18 (4.6 · 250 mm, 5 lm)
reverse phase column on an HP-Series 1100 HPLC (Agi-
lent Corp, Palo Alto, CA, USA) with a linear gradient
of 0–100% methanol in 0.1% TFA/water.
4.7. Effects of metal ions and inhibitors on enzyme activity
TLC of hydrolyzed products was performed on
analytical silica gel 60 F254 aluminum plates (Merck,
Darmstadt, Germany) with CHCl3/MeOH/H2O (15:3:1)
as solvent.
The effects of some metal ions and inhibitors on activ-
ity were determined by pre-incubating the purified en-
zyme with the substance for 10 min at 30 ꢁC in 0.1 M
sodium acetate, pH 5.0, then adding 1 mM of pNP-b-
D-fucoside or 2 mM pNP-b-D-glucoside and incubating
for 10 min at 30 ꢁC. For d-gluconolactone, assays were
done without preincubation and the inhibitor was
freshly prepared, since it is susceptible to break-down
in water (Combes and Birch, 1988).
4.10. Sugar analysis
Glycoside (5.5 mg) was boiled with 0.7 mL 0.1 N
H2SO4 in a 1.5 mL microcentrifuge tube for 25 min.
The solution was cooled and extracted with n-butanol,

1888
P. Chuankhayan et al. / Phytochemistry 66 (2005) 1880–1889
Adinaraya, D., Rajasekhara Rao, J., 1975. Isoflavonoids of Dalbergia
paniculata. Indian J. Chem. 13, 425–426.
Babcock, G.D., Esen, A., 1994. Substrate specificity of maize b-
glucosidase. Plant Sci. 101, 31–39.
Barrett, T., Suresh, C.G., Tolley, S.P., Dodson, E.J., Hughes,
M.A., 1995. The crystal structure of a cyanogenic b-glucosidase
from white clover, a family 1 glycosyl hydrolase. Structure 3,
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then neutralized with NaHCO3 and the aqueous layer
was dried by rotary evaporator. The trimethylsilylation
of sugar was done by the method of Nikolov and Reilly
(1983) with three milligrams of dried residue. The trim-
ethylsilylation mixture was injected into the gas
chromatograph and separated with the following condi-
tions: injector at 250 ꢁC; split ratio 20:1; helium carrier
gas at 1.2 mL/min; the column temperature was held at
150 ꢁC for 5 min and increased to 190 ꢁC at 20 ꢁC/min
and held for 20 min. Mass spectra were collected with a
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4.11. Compound 1, dalpatein-7-O-[b-D-apiofuranosyl-
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Compound 1 was obtained as an amorphous light
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nm: 260 and 315; IR (KBr) vmax cmꢀ1: 3435 (OH),
1709, 1639, 1559, 1412, 1270, 795, 639; For 1H and
13C NMR spectra, see Table 1. FT-MS m/z 659
[M + Na]+ calculated for C29O16H32.
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3436 (OH), 1706, 1639, 1561, 1412, 808, 643, 524; For
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Acknowledgements
The authors gratefully acknowledge the assistance
and advice of Phayungsak Tanglukmongkol (Bruker,
Thailand) for mass spectrometric analysis of substrates
and Dr. Chantragan Srisomsap for protein sequencing.
Sunaree Rimlumduan is thanked for assistance in sub-
strate preparation. Prof. Atsuo Kimura, Dr. Eri Fuku-
shi, Dr. Palangpon Kongsaeree, Dr. Prasart Kittipoop
and Dr. Adrian Flood are thanked for advice on struc-
tural characterization. P.C. is a Royal Golden Jubilee
Ph.D. Fellow and J.S. is a Senior Research Fellow of
the Thailand Research Fund. This work was supported
by grants from Suranaree University of Technology
and the Thailand Research Fund (BRG4780020).
Rajasekhara Rao, J., Srinivasa Rao, R., 1991. Dalpaniculin, A C-
glycosylisoflavone from Dalbergia paniculata seeds. Phytochemistry
30, 715–716.
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