Biotransformation of daidzein to equol by crude enzyme from Asaccharobacter celatus AHU1763 required an anaerobic environment

Article abstract of DOI:10.1271/bbb.80908

Asaccharobacter celatus AHU1763 is a Gram-positive, obligate anaerobic, non-spore forming, rod-shaped bacteria that was successfully isolated from rat cecal content. Daizein was converted to equol via dihydrodaidzein by this bacterium. A crude enzyme that converted daidzein to dihydrodaidzein was detected mainly in the culture supernatant. The ability of this enzyme dropped after the culture supernatant was exposed to a normal atmospheric environment for even 5 min. Furthermore, the enzyme responsible for changing dihydrodaidzein to equol was detected mainly in the cell debris, which required anaerobic conditions for its activity.

Full text of DOI:10.1271/bbb.80908

Biosci. Biotechnol. Biochem., 73 (6), 1435–1438, 2009
Note
Biotransformation of Daidzein to Equol by Crude Enzyme
from Asaccharobacter celatus AHU1763 Required an Anaerobic Environment
y
Charin THAWORNKUNO, Michiko TANAKA, Teruo SONE, and Kozo ASANO
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Kita 9, Nishi 9, Kita-Ku, Sapporo, Hokkaido 060-8589, Japan
Received December 24, 2008; Accepted February 23, 2009; Online Publication, June 7, 2009
[doi:10.1271/bbb.80908]
Asaccharobacter celatus AHU1763 is a Gram-positive,
intestinal microbiota of such individuals are stable.
Therefore, non-equol producers excrete no EQL, even
when they ingest soy protein powder (34 g/d) for 1
obligate anaerobic, non-spore forming, rod-shaped
bacteria that was successfully isolated from rat cecal
content. Daizein was converted to equol via dihydro-
daidzein by this bacterium. A crude enzyme that
converted daidzein to dihydrodaidzein was detected
mainly in the culture supernatant. The ability of this
enzyme dropped after the culture supernatant was
exposed to a normal atmospheric environment for even
1
0)
month.
There are few reports on daidzein-metabolizing
intestinal bacteria. An anaerobic gram-positive strain,
1
1)
HGH 6, that converts DAI to DHD, and Clostridium
sp. strain HGH 136, and Eubacterium ramulus,13) that
convert DAI to O-DMA, were isolated from humans.
The human intestinal bacterium SNU-Julong 732 that
12)
5
min. Furthermore, the enzyme responsible for chang-
1
4)
ing dihydrodaidzein to equol was detected mainly in the
cell debris, which required anaerobic conditions for its
activity.
converts DHD to EQL was also isolated from human.
A mixture of Lactobacillus mucosae, Enterococcus
1
5)
faecium, Finegoldia magna, and Veillonella sp.
produces EQL from daidzein. However, only some
Key words: Asaccharobacter celatus AHU1763; daid-
zein; dihydrodaidzein; equol
strains, Asaccharobacter celatus AHU1763 (strain
1
6)
do03),
1
Lactococcus 20-92 isolated from human
18)
7)
feces,
and Adlercreutzia equolifaciens,
directly
Phytoestrogens are estrogen-like compounds found in
a wide variety of plants. There are three major classes of
phytoestrogens: isoflavones, lignans, and coumestans.
They are classified as phytoestrogens because their
structures resemble the mammalian estrogen (17ꢀ-
estradiol) and they have a weak affinity for the estrogen
receptor.1 Epidemiologic and experimental studies
showed that phytoestrogen has potentially preventive
effects on breast cancer, prostate cancer, cardiovascular
produce EQL from DAI as sole strain.
A. celatus AHU1763 was isolated from rat cecal
1
9)
content by our lab. Current evidence for the con-
version of DAI to EQL in bacteria is unavailable. Our
study is the first to determine the enzymatic reaction
involved in the conversion reaction of DAI to EQL. We
investigated certain parameters that might affect the
ability of the enzyme, such as assayed and storage
conditions, including temperature and pH values.
)
2
,3)
disease, osteoporosis, and menopausal symptoms.
Soy-based food is the predominant food source of
isoflavones. Among isoflavones, daidzein (DAI) and
A. celatus AHU1763 was cultured anaerobically on
GAM medium (Nissui Pharmaceutical, Tokyo) supple-
mented with 1% arginine (Wako, Tokyo) and 200 mM
4
)
ꢀ
genistein (GEN) are the most intensively studied. In
plants, most isoflavones exist as glycosides, mainly as
daidzin and genistin. They are biologically inactive;
once ingested, they are cleaved by ꢀ-glucosidase to
aglycone, DAI, and GEN. By intestinal microflora, DAI
is converted either to equol (EQL) or O-desmethylan-
golensin (O-DMA) via dihydrodaidzein (DHD), while
the metabolism of GEN in humans is not well
characterized. The half-life in the body for EQL is
significantly longer than DAI or GEN,5 and EQL is
consistently reported to be present at high levels in the
blood.6 In addition, EQL has stronger estrogenic
DAI (LC Laboratory, Massachusetts) at 37 C in an
anaerobic chamber (Coy Laboratory Products, Michigan)
under 85% N2, 10% CO2, 5% H2. After 6 h of
cultivation, the cell entered the log-phase of growth.
Then they entered the stationary phase of growth after
9 h of cultivation (Fig. 1A). Isoflavone concentrations in
culture broth during the growth of A. celatus AHU1763
were measured using HPLC. Aliquots of the assay were
extracted 3 times with ethyl acetate of 1.5 volume (of
the sample), and then evaporated using a rotary
evaporator. After that, aliquots were dissolved in
methanol and filtered using a 0.45-mm filter (Millipore,
Massachusetts). Each sample was injected into HLPC
(Jasco, Tokyo) equipped with a Mightysil RP-18 GP
(3:0 ꢁ 250 mm; 3 mm, Kanto chemical, Tokyo) and a
UV detector (280 nm; Jasco). The mobile phase was
a solution of water: acetonitrile: acetic acid, 85:15:0.1
)
)
7
,8)
activity than DAI or O-DMA.
Humans capable of
producing EQL from DAI (equol producers) have a
lower risk of developing breast and prostate cancers than
9
)
non-equol producers. Daidzein-metabolizing pheno-
9
)
types are stable in individuals over time, because the
y
To whom correspondence should be addressed. Tel: +81-11-706-2493; Fax: +81-11-706-4961; E-mail: asanok@chem.agr.hokudai.ac.jp
Abbreviations: DAI, daidzein; GEN, genistein; EQL, equol; O-DMA, O-desmethylangolensin; DHD, dihydrodaidzein; PLP, pyridoxal
-phosphate; DTT, dithiothreitol
0
5

1436
C. THAWORNKUNO et al.
Table 1. The Effect of Coenzyme on Production of Daidzein
Derivatives
A
10
Isoflavone concentration (mM)
DAI DHD EQL
Coenzyme
1
Culture supernatant No coenzyme 81:4 ꢂ 0:9 14:7 ꢂ 0:7 43:0 ꢂ 0:9
ATP
NAD
80:5 ꢂ 0:8 15:5 ꢂ 0:2 43:3 ꢂ 0:8
84:1 ꢂ 2:3 15:8 ꢂ 0:5 43:3 ꢂ 0:5
83:2 ꢂ 1:4 15:8 ꢂ 0:4 42:6 ꢂ 0:4
78:3 ꢂ 1:3 22:1 ꢂ 1:2 42:8 ꢂ 0:6
69:6 ꢂ 1:0 27:5 ꢂ 3:3 43:2 ꢂ 0:2
84:5 ꢂ 2:9 16:9 ꢂ 1:5 41:3 ꢂ 0:7
NADH
NADP
NADPH
PLP
0
.1
0
.01
0
6
12
18
Cell debris
No coenzyme 9:5 ꢂ 0:1 57:7 ꢂ 2:5 31:1 ꢂ 0:4
ATP
NAD
8:9 ꢂ 0:2 59:4 ꢂ 0:7 26:9 ꢂ 0:1
10:3 ꢂ 0:4 57:3 ꢂ 0:6 32:3 ꢂ 1:0
11:0 ꢂ 0:7 50:6 ꢂ 1:1 36:3 ꢂ 2:2
10:9 ꢂ 0:9 76:4 ꢂ 1:6 11:5 ꢂ 0:3
0 28:6 ꢂ 0:7 70:9 ꢂ 0:7
Incubation time (h)
NADH
NADP
NADPH
PLP
B
2
00
50
00
8:5 ꢂ 0:5 71:8 ꢂ 0:6 18:1 ꢂ 0:5
1
Values are isoflavone concentrations (mean ꢂ SD of 3 values). Culture
supernatant was used as a crude enzyme to determine the change of DAI to
DHD. Cell debris was used as a crude enzyme to determine the change of
DHD to EQL. Coenzyme (0.2 mM) was used for each reaction. The amount
of protein contained in the assay mixture was 35.7 and 16.7 mg/ml for
culture supernatant and cell debris respectively.
1
50
0
responsible for changing DAI to DHD, each crude
ꢀ
0
6
12
18
enzyme was incubated in an anaerobic chamber at 37 C
for 16 h in a reaction mixture containing 50 mM MOPS-
NaOH pH 7.0, 0.2 mM NADPH, 1 mM valinomycin, and
100 mM DAI. Isoflavone concentrations in each assay
reaction were measured using HPLC, as described above.
Change of DAI to DHD was detected when the culture
supernatant was used as a crude enzyme, and some
change was also detected in cell extract and cell debris
(data not shown). The enzyme responsible for changing
DHD to EQL, each crude enzyme was incubated under
the same conditions as described above in a reaction
mixture containing 50 mM MOPS-NaOH pH 7.0, 0.2 mM
NADPH, 1 mM valinomycin, and 100 mM DHD. Change
of DHD to EQL was detected when cell debris was used
as a crude enzyme, and some change was also detected
from the cell extract (data not shown).
Incubation time (h)
Fig. 1. Isoflavone Concentration Profile during the Growth of
ꢀ
A. celatus AHU1763 at 37 C for 18 h.
The growth of A. celatus AHU1763 was plotted between the
incubation time and growth (log OD600) (A). The isoflavone
concentration including DAI ( ), DHD ( ), and EQL ( ) was
measured during the growth of A. celatus AHU1763 and plotted as
the unit of mM (B).
(
V/V/V), the flow rate was 0.4 ml/min, and the column
ꢀ
temperature was 60 C. Metabolites were identified by
comparing their retention times with those of standards.
DAI rapidly decreased during the late-lag phase of
growth (5 h after cultivation), in a culture broth of
A. celatus AHU1763 (Fig. 1B). The reduction of DAI
resulted in the accumulation of DAI metabolites (DHD
and EQL).
þ
þ
Many coenzymes, ATP, NAD , NADH, NADP ,
0
NADPH, and pyridoxal 5 -phosphate (PLP), were used
to determine the effect on enzyme ability. The enzyme
responsible for changing DAI to DHD, the culture
supernatant was incubated in an anaerobic chamber at
All steps of crude enzyme preparation were handled
under strictly anaerobic conditions. A. celatus AHU1763
was cultured anaerobically on GAM medium supple-
mented with 1% arginine and 100 mM DAI. When the
cells entered the stationary phase, the bacterial culture
broth was centrifuged and cells were separated. Then
harvested culture supernatant was filtered through a 0.22-
mm filter. The separated cell was washed 3 times and
resuspended with sterile distilled water. Valinomycin
was added to the cell suspension at a final concentration
of 1 mM, in order to kill the living cells. The cells in
suspension were disrupted by bead shocker. Bacterial
cell extract and cell debris were separated by centrifu-
gation at 18,000 g for 20 min. Then the cell extract was
filtered through a 0.22-mm filter. The cell debris was
washed 3 times, and then it was resuspended in sterile
distilled water with 1 mM of valinomycin. Finally, crude
enzyme from the culture supernatant, cell extract, and
cell debris was obtained. To identify the enzyme
ꢀ
37 C in reaction mixtures containing 50 mM MOPS-
NaOH pH 7.0, 0.2 mM coenzyme, and 100 mM DAI.
When compared to no coenzyme addition, the DAI
concentration decreased 3.1 and 11.8 mM, while the DHD
þ
concentration increased 7.4 and 12.8 mM, when NADP
and NADPH were used respectively. For the enzyme
responsible for changing DHD to EQL, cell debris was
ꢀ
incubated in an anaerobic chamber at 37 C in reaction
mixtures containing 50 mM MOPS-NaOH pH 7.0,
0.2 mM coenzyme, 1 mM valinomycin, and 100 mM
DHD. When compared to no coenzyme addition, the
DHD concentration decreased 29.1 mM, while the EQL
concentration increased 39.8 mM when NADPH was
used (Table 1). Because, NADPH plays a role in the
redox reaction, which belongs to oxidoreductase group
of enzymes. A change of DAI to EQL via DHD requires
NADPH. It is suggested that the enzymatic reactions

Biotransformation of Daidzein to Equol by A. celatus AHU1763
1437
A
B
1
1
1
40
20
00
120
1
00
80
60
40
20
0
80
60
40
20
0
4
5
6
7
8
9
10
4
5
6
7
8
9
10
pH
pH
C
D
1
1
60
20
240
2
1
00
60
8
0
0
0
120
8
0
0
0
4
4
2
5
30
35
40
45
25
30
35
40
45
Incubation temperature (° C)
Incubation temperature (° C)
Fig. 2. Effect of pH and Temperature on Enzyme Ability.
The ability were measured at various pHs (supernatant, A; and cell debris, B) with three buffer systems: 50 mM Citrate-NaOH, pH 4 to 6;
ꢀ
50 mM MOPS-NaOH, pH 7 to 8; and 50 mM Glycine-NaOH, pH 9 to 10 after incubation in an anaerobic chamber at 37 C for 16 h, at various
temperatures (supernatant, C; and cell debris, D), from 25 to 45 C incubated in a 50 mM MOPS-NaOH, pH 7.0 buffer system for 16 h. Each
ꢀ
point represents the concentration of isoflavone production as percentage ꢂ SD of three values. % Relative production at each point was
ꢀ
compared to production of isoflavone at pH 7 at 37 C. The amount of protein contained in the assay mixture was 35.7 and 16.7 mg/ml for the
culture supernatant and cell debris respectively.
involved in this pathway belong to the oxidoreductase
group of enzymes. DAI is converted to DHD by
hydrogenation between the C-2 and C-3 positions. The
keto group of DHD at C-4 position is converted to the
alcohol group to yield 4-hydroxy-equol. Then it is
converted to EQL by removal of the alcohol group at the
C-4 position. However, we could not detect the 4-
hydroxy-equol in the chromatogram. Perhaps, this
compound is not stable and rapidly changed to equol
or was high in the turnover number of the enzyme that
changes 4-hydroxy-equol to EQL.
Because A. celatus AHU1763 is an obligate anaerobic
bacterium, we investigated to determine whether crude
enzyme has ability for the conversion of DAI to EQL
even under aerobic conditions. Culture supernatant and
cell debris were used as a crude enzyme to assay for the
conversion of DAI to DHD and DHD to EQL respec-
tively. DHD increased 12.3 and 0.1 mM, when the culture
supernatant was assayed under anaerobic and aerobic
condition respectively. EQL increased 70.1 and 0.6 mM
when the cell debris was assayed under anaerobic and
aerobic conditions respectively. Change of DAI to DHD
and DHD to EQL dramatically decreased when the
enzymes were assayed under aerobic conditions. These
results suggest that enzymes should be assayed under
anaerobic conditions.
ed above. DHD increased 3.7, 6.0, 9.8, and 10.7 mM after
the culture supernatant was stored at 37, 4, ꢃ20, and
ꢀ
ꢃ80 C respectively. EQL increased 0, 10.2, 65.3, and
69.0 mM after the cell debris was stored at 37, 4, ꢃ20,
ꢀ
and ꢃ80 C respectively. In addition, crude enzymes
were carried out from an anaerobic chamber and
vigorously shaken in a normal aerobic atmospheric
environment for 5 min before assay under anaerobic
conditions. Ability to change DAI to DHD of the culture
supernatant dropped dramatically and DHD did not
increase, as compared to the non-exposed condition.
Furthermore, strong biochemical reducing agents such
as dithiothreitol (DTT), ꢀ-mercaptoethanol, and ascorbic
acid at final concentrations of 2 mM, 0.5% V/V, and
1 mM respectively, were added to the reaction mixture
before exposure to a normal atmospheric environment.
No increase in the DHD concentration could be obtained
(data not shown), while EQL increased 65.1 mM after the
cell debris was exposed to a normal atmospheric
environment.
The ability in changing DAI to DHD and DHD to
EQL was measured at various pHs (4.0 to 10.0) and
ꢀ
temperatures (25 to 45 C) (Fig. 2). The ability in
changing DAI to DHD form the culture supernatant
increased from pH 4.0 and reached a maximum at
pH 6.0, when the culture supernatant was incubated at
ꢀ
In order to determine the effects of storage conditions
of crude enzymes, the ability in changing isoflavone
after storage at different conditions was studied. Crude
enzymes from culture supernatant and cell debris were
tightly capped to maintain an anaerobic environment.
They were stored at different temperatures, 37, 4, ꢃ20,
37 C for 16 h (Fig. 2A), while the ability in changing
DHD to EQL from cells debris increased from pH 4.0
and reached a maximum at pH 7.0 (Fig. 2B).Changing
of isoflavone at various temperatures when incubated in
a 50 mM MOPS-NaOH, pH 7.0 buffer system is shown
in Fig. 2C and D. The highest enzyme ability was
obtained from incubating the culture supernatant and
ꢀ
and ꢃ80 C, for 16 h. Then the crude enzymes were
ꢀ
assayed under an anaerobic environment as the describ-
cell debris at 30 C.

1438
C. THAWORNKUNO et al.
In summary, as shown in the results above, DAI was
6) Rowland IR, Wiseman H, Sanders TA, Adlercreutz H, and
Bowey EA, Nutr. Cancer, 36, 27–32 (2000).
changed to EQL via DHD at the early stage of growth of
A. celatus AHU1763. To study enzymes characteristics,
crude enzymes were harvested. Enzymes responsible
for changing DAI to DHD and DHD to EQL were
located mainly in the culture supernatant and cell debris
respectively. Both enzymes responsible for changing
DAI to DHD and DHD to EQL should be assayed under
anaerobic conditions, which require NADPH for the
reaction. In addition, crude enzymes from both the
culture supernatant and cell debris were exposed to a
normal atmospheric environment. A drastic reduction in
enzyme ability was observed after the culture super-
natant was exposed to a normal atmospheric environ-
ment, while the enzyme ability of the cell debris was not
significantly decreased.
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5