Pathways of biotransformation of zingiberen newsaponin from Dioscorea zingiberensis C. H. Wright to diosgenin

Article abstract of DOI:10.1016/j.molcatb.2013.09.013

A new steroidal saponin-β-glucosidase from Aspergillus flavus that specifically hydrolyzed the terminal β-d-glucosyl group at the C-3 position of zingiberen newsaponin, deltonin and trillin from Dioscorea zingiberensis C. H. Wright (DZW) was purified, and characterized. The optimal temperature and pH for the new steroidal saponin-β-glucosidase was 50 C and pH 5.0. The steroidal saponin-β-glucosidase was stable at 30-60 C, and retained more than 80% activity. Further, the purified protein was analyzed by ESI-Q-TOF proteomic analyzer. The results indicated that this enzyme is a β-glucosidase of the type glycosidase hydrolase 3 (GH3). Using a combination of the steroidal saponin-β-glucosidase and steroidal saponin-α-1,2-rhamnosidase from Curvularia lunata obtained previously in our lab, the saponins zingieren newsaponin and deltonin could be converted to diosgenin. The pathways of converting zingiberen newsaponin and deltonin into diosgenin by the two key enzymes were elucidated in this study.

Full text of DOI:10.1016/j.molcatb.2013.09.013

Journal of Molecular Catalysis B: Enzymatic 98 (2013) 1–7
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Pathways of biotransformation of zingiberen newsaponin from
Dioscorea zingiberensis C. H. Wright to diosgenin
a,b,1
a,b,1
b
a
a
a
Hongzhi Huang
, Min Zhao
, Li Lu , Dawei Tan , Wenbin Zhou , Chengqi Xiong ,
a
b
c
a,∗
Yang Zhao , Xinbo Song , Liyan Yu , Baiping Ma
Beijing Institute of Radiation Medicine, No. 27, Taiping Road, Beijing 100850, PR China
Tianjin University of Traditional Chinese Medicine, No. 88, Yuquan Road, Tianjin 300193, PR China
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 March 2013
Received in revised form 30 July 2013
Accepted 7 September 2013
Available online 16 September 2013
A new steroidal saponin-␤-glucosidase from Aspergillus flavus that specifically hydrolyzed the terminal
␤-d-glucosyl group at the C-3 position of zingiberen newsaponin, deltonin and trillin from Dioscorea
zingiberensis C. H. Wright (DZW) was purified, and characterized. The optimal temperature and pH for
◦
the new steroidal saponin-␤-glucosidase was 50 C and pH 5.0. The steroidal saponin-␤-glucosidase
◦
was stable at 30–60 C, and retained more than 80% activity. Further, the purified protein was ana-
lyzed by ESI-Q-TOF proteomic analyzer. The results indicated that this enzyme is a ␤-glucosidase of
the type glycosidase hydrolase 3 (GH3). Using a combination of the steroidal saponin-␤-glucosidase and
steroidal saponin-␣-1,2-rhamnosidase from Curvularia lunata obtained previously in our lab, the saponins
zingieren newsaponin and deltonin could be converted to diosgenin. The pathways of converting zin-
giberen newsaponin and deltonin into diosgenin by the two key enzymes were elucidated in this study.
Keywords:
Biotransformation
Zingiberen newsaponin
Steroidal saponin-␤-glucosidase
Diosgenin
Pathways
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
the serious environmental problems [7]. With the development
of biotechnology, mild, safe and environmentally friendly meth-
ods are being applied to biotransformation of more and more
natural products. In recent years, enzymatic productions of dios-
genin from DZW have been reported. Some saponin glycosidases
were extracted and purified from microorganisms, plants and liv-
ers. The dioscin-␣-l-rhamnosidase from pig liver [8] hydrolyzes
terminal 1,2-linked rhamnosyl and 1,4-linked rhamnosyl from
dioscin to 3-O-␤-D-Glc-diosgenin. The dioscin-glycosidase from
Absidia sp.d38 [9] gradually hydrolyzes terminal 1,2 linked rham-
nosyl, 1,4 linked rhamnosyl and 3-O-␤-d-glucosyl from dioscin to
diosgenin as final product. In our lab, related reports have been
published. Feng [10,11] found that a glucoamylase from Curvularia
lunata has steroidal saponin–rhamnosidase activity. The enzyme
can hydrolyze the terminal ␣-1,2-rhamnosyl residue at the C-3
position of dioscin. Very recently, Lei [12] purified a ␤-glucosidase
from Aspergillus fumigates that can hydrolyze DZW spirostano-
sides, such as trillin, diosgenin-diglucoside, dioscin, deltonin and
gracillin, to produce diosgenin. However, the pathway of the trans-
formation process had not been elucidated. Dong [13] presumes
the pathways of biotransformation of DZW by Aspergillus oryzae,
but do not purify the key enzymes to confirm the pathways.
In this study, we purified and identified a ␤-glucosidase
from Aspergillus flavus that could hydrolyze terminal 1,4-linked
glucosyl residue from zingiberen newsaponin and terminal
Diosgenin (25R-spirost-en-3␤-ol), an aglycon of variety of
steroidal saponins in plants, is an important precursor for syn-
thesizing steroidal hormones and steroidal contraceptives [1].
Recently, there are many reports on the significant pharmaco-
logical attributes of diosgenin, such as antagonistic effect on
cardiovascular diseases [2], anticancer activity, anti-skin aging
and anti-hepatitis C virus [3–5]. Diosgenin mainly exists in the
plants rhizomes as steroidal saponins which are its glycosides
with different sugar groups [6]. In China, Dioscorea zingiberen-
sis C. H. Wright (DZW) is the preferred species as the resources
of its raw material. In industry, diosgenin is produced by strong
acid treatment of the DZW rhizomes. However, a great deal of
wastewater generated during the production process leads to
∗ Corresponding author at: Beijing Institute of Radiation Medicine, 27# Tai-ping
Road, Haidian District, Beijing 100850, China. Tel.: +86 10 68210077x930265;
fax: +86 10 68214653.
E-mail addresses: hongzhih@gmail.com (H. Huang), zhao06min05@aliyun.com
(M. Zhao), comic-3344@163.com (L. Lu), tandawei1981@163.com (D. Tan),
zwb1983114@163.com (W. Zhou), Xiongchengqi2005@sohu.com (C. Xiong),
mmzhaoy@163.com (Y. Zhao), songxinbo@tjutcm.edu.cn (X. Song),
yuliyan 2000@yahoo.com (L. Yu), mabaiping@sina.com (B. Ma).
1
These two authors contributed equally to this study.
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.09.013

2
H. Huang et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 1–7
1
,3-linked glucosyl residue from deltonin to prosapogenin A of
2.5. Enzyme purification
dioscin. Using a combination of the ␤-glucosidase and steroidal
saponin-␣-1,2-rhamnosidase from C. lunata obtained previously in
our lab, the saponins zingiberen newsaponin and deltonin could
be converted into diosgenin step by step, so that the pathways of
biotransformation of zingiberen newsaponin into diosgenin by the
two key enzymes were elucidated for the first time.
◦
All column operations were conducted at 25 C. The collected
fractions were tested for enzyme activity, and zingiberen news-
aponin solution (3.0 mg/mL) was used as substrate.
The crude enzyme was precipitated by solid ammonium sulfate
(80% saturation) and stored at 4 C overnight. The protein pre-
◦
cipitated was collected by centrifuging (12,000 rpm/min, 15 min),
then dissolved and dialyzed against the 25 mM Tris–HCl buffer,
pH 6.0. The dialysate was loaded on a column of QFF which was
pre-equilibrated with 25 mM Tris–HCl buffer (pH 6.0) and then
eluted with a gradient of 0–1.0 M NaCl in the same buffer at
2
. Materials and methods
2.1. Materials
4
.0 mL/min. The flow-through fractions which showed high zin-
Zingiberen newsaponin, deltonin, prosapogenin A of dioscin,
giberen newsaponin hydrolysis activity were collected. The active
proteins were further purified on a column of SFF equilibrated
with 25 mM citric acid buffer, pH 4.5, then eluted with the same
buffer in a linear gradient of 0–1.0 M NaCl. The flow rate was set as
trillin and diosgenin were prepared by our laboratory. A. oryzae
.4437, Aspergillus niger 3.7390, A. flavus 3.2792, Penicillium
3
purpurogenum 3.5160 and Mucor wutungkiao 3.0025 were pur-
chased from Institute of Microbiology Chinese Academy of
Sciences. Middle molecular weight marker proteins for sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
were obtained from Fermentas (Amercia). Coomassie brilliant blue
R-250 was obtained from Serva (Heideberg, Germany). S-Sepharose
Fast Flow (SFF) and Q-Sepharose Fast Flow (QFF) were purchased
from Amersham Biosciences (GE Healthcare Co. Ltd., Chalfont St
Giles, UK). High-performace liquid chromatography (HPLC) analy-
sis was carried out on Waters 2695 unit with Alltech Evaporative
Light Scattering Detector (ELSD) 2000 using an Agilent TC-C18
2
.0 mL/min. The homogeneity of the active fraction was checked by
SDS-PAGE.
2.6. Catalytic characteristics and analytic methods of the purified
enzyme
All activities surveyed for characterization were done on zin-
giberen newsaponin (3.0 mg/mL) as substrate, and measured the
increase of diosgenin by HPLC analysis. The optimum pH was tested
(
4.6 mm × 250 mm, 5 ␮m). Thin-layer chromatography (TLC) anal-
◦
at 50 C in 0.2 M Na HPO –citric acid buffer at pH range 3.0–8.0
ysis was carried out on precoated silica gel GF2 plates (0.25 mm
2
4
54
for 24 h. The optimum temperature was determined from 30 to
thick, Qindao Haiyang Chemical Group, China). Visualization of the
◦
7
0 C at the optimum pH for 24 h. Thermostability was measured
TLC plates was performed by 10% H SO –EtOH spray reagent, fol-
2
4
by preincubation of the enzyme at the optimum pH at differ-
lowed by heating. All the other chemicals used were of analytical
grade, and solvents for HPLC were chromatography grade.
◦
ent temperatures from 30 to 70 C for 1 h, and then co-incubated
with substrate solution for 24 h. The optimum reaction time was
examined from 24 to 144 h at the optimum temperature and pH
value.
2.2. Microorganism cultivating
The five microorganisms, respectively, were rejuvenated on the
potato dextrose agar (PDA) slant for 3 to 7 days before being used
in transformation experiments, and then grow in the medium con-
taining corn steep liquor (1.5%), glucose (1.0%), peptone (1.0%), and
2
2
.7. Biotransformation and purification of saponins
.7.1. Biotransformation
Biotransformation of zingiberen newsaponin and deltonin to
◦
yeast extract (0.5%), pH 6.0 at 25 ± 1 C, 150 rpm/min for 5 days in
a constant temperature shaker.
prosapogenin A of dioscin by the purified enzymes were carried
out in 250 mL conical flasks each containing 300 mg zingiberen
newsaponin, 200 mg deltonin and 15 mL purified enzyme solution
in 0.2 M Na HPO –citric acid buffer (pH 5.0), and total volume
is 100 mL. The culture was carried out at 50 C with shaking
at 150 rpm. Samples were harvested by extracted with equal
2.3. Crude enzyme extraction
2
4
The 7-day-old PDA slant was used to incubate at 500 mL erlen-
◦
meyer flask which containing 200 mL of sterilized liquid medium.
The flasks were incubated on a rotary shaker at 150–180 rpm/min
◦
volume of water-saturated n-butanol for three times at 25 C,
◦
at 25 ± 1 C for 5 days, and then centrifuged to remove mycelia and
the n-butanol layer was collected, concentrated and freeze-dried,
then the transformed products were harvested. Biotransforma-
tion of prosapogenin A of dioscin to trillin by the steroidal
saponin-␣-1,2-rhamnosidase and conversion of trillin to diosgenin
by the purified enzyme were carried out as the above proce-
dure.
the supernatant was used to further experiment as crude enzyme.
2.4. Screening of microorganisms
Both zingiberen newsaponin and deltonin were soluted by dis-
tilled water, and the final concentration was 3.0 mg/mL, followed
by sonication for make the solution suspended for three minutes.
One hundred microliters of substrate solution was added into
2
.7.2. Purification of saponins
The crude converted products were dissolved into methanol
1
1
25 ␮L of 0.2 M Na HPO –citric acid buffer (pH 5.0), and then
2 4
00 ␮L of crude enzyme was added into the system for reac-
and then isolated passed through normal-phase silica gel col-
umn (␸ 1.5 cm, L 20 cm) and eluted with CHCl –MeOH–H O
◦
tion. After incubation at 50 C for 24 h, n-butanol was added
to stop the reaction. The transformed product (n-Butanol layer)
3
2
(60:10:0.01, 12:1:0.01, 18:1:0.01) to get prosapogenin A of
was ascertained by TLC using CHCl /MeOH/H O = 65:35:10 (v/v)
3
2
dioscin, trillin, diosgenin, respectively. All the compounds obtained
were further purified by crystallization to a purity of over 95%
(determined by HPLC-ELSD). These compounds were used as
standards for HPLC analysis and substrates for biotransforma-
tion.
as developing solvent. Visualization was performed by 10%
H SO –EtOH spray reagent, followed by heating. The solvent
2
4
◦
system of HPLC was MeOH/H O (gradient), temperature 90 C, flow
2
rate 1.0 mL/min, and gas-flow rate 2.4 L/min.

H. Huang et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 1–7
3
Fig. 1. Results of TLC analysis using five microorganisms. (a) Biotransformation of zingiberen newsaponin. (b) Biotransformation of deltonin. S1, zingiberen newsaponin,
S2, deltonin, Ao, Aspergillus oryzae. Af, Aspergillus flavus. S3, Prosapogenin A of dioscin. An, Aspergillus niger. Mw, Mucor wutungkiao. Pp, Penicillium purpurogenum. The
converted products were ascertained by thin-layer chromatography (TLC) on silica gel GF254 plate using CHCl3/CH3OH/H2O = 65:35:10 as developing solvent. Visualization
was performed by 10% H2SO4–EtOH spray reagent, followed by heating.
3
. Results and discussion
terminal 1,2-linked rhamnose group was quickly hydrolyzed by
rhamnosidase from A. oryzae. Our results indicated, however, that
while the terminal glucose groups of the two saponins were
hydrolyzed in the broths, meanwhile the rhamnose group still
remained. In our research, the supernatants of the fermentation
broths of the fungi were used for the biotransformation of zin-
giberen newsaponin and deltonin. No inducers were added into
the cultures of the fungi. This could be the reason leading to the
differences between other reports and our study.
3.1. Screening of fungi for transforming zingiberen newsaponin
and deltonin
Five fungi A. oryzae (Ao), A. niger (An), A. flavus (Af), P. pur-
purogenum (Pp) and M. wutungkiao (Mw) were selected, and their
culture broths were used for transforming zingiberen newsaponin
and deltonin. According to the TLC analytical results, the culture
broth of A. flavus showed the best performance on hydrolyzing zin-
giberen newsaponin to deltonin and prosapogenin A of dioscin,
and hydrolyzing deltonin to prosapogenin A of dioscin. A. oryzae
showed weak activity on hydrolyzing zingiberen newsaponin to
deltonin (Fig. 1). Further, the products of zingiberen newsaponin
and deltonin converted by A. flavus broth were confirmed by HPLC
In order to figure out what enzyme in the broth of A. flavus con-
tributed to the biotansformation, the crude enzymes were purified
and identified.
3.2. Enzyme purification and converting zingiberen newsaponin
and deltonin into prosapogenin A of dioscin
(
Fig. 2).
In the previous report [13], both zingiberen newsaponin and
deltonin were used as inducers cultured with A. oryzae, and the
The enzyme was purified using the method of zingiberen news-
aponin activity guide to homogeneity from culture broth of A.
Fig. 2. HPLC chromatographs of biotransformation of zingiberen newsaponin and deltonin by A. flavus. (a) Zingiberen newsaponin. (b) Deltonin. (c) Prosapogenin A of dioscin.
d) Bioransformed products of deltonin. (e) Biotransformed products of zingiberen newsaponin.
(

4
H. Huang et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 1–7
◦
Fig. 5. The optimal pH of steroidal saponin-␤-glucosidase (50 C, 0.2
M
Na2HPO4–citric acid buffer).
flavus. The crude enzyme solution was purified by anion chro-
matography (QFF) and cation chromatography (SFF), and a single
band on SDS-PAGE was obtained (Fig. 3). The purified enzyme
showed activity to hydrolyze zingiberen newsaponin and deltonin
into prosapogenin A of dioscin (Fig. 4). Based on the substrates
of the enzyme, it was defined as steroidal saponin-␤-glucosidase.
From Fig. 3, the molecular weight of the enzyme was about
1
20 kDa.
3.3. Identification of the purified enzyme
The purified enzyme band on SDS-PAGE was submitted to Bei-
jing Proteome Research Center for protein sequencing. After the
protein was digested with trypsin and the peptides were separated
by the ESI-Q-TOF, 10 peptides were obtained and sequenced, as
shown in Table 1. A blast search of these sequences was performed
in National Center for Biotechnology Information database – Basic
Local Alignment Search Tool (NCBI-BLAST), and the sequences dis-
played the highest similarity to the ␤-glucosidase A (EC 3.2.1.21)
from A. oryzae (Table 2).
Fig. 3. SDS – polyacrylamide gel electrophoresis. Lane 1, molecular weight makers:
␤
-galactosidase (120 kDa), bovine serum albumin (85 kDa), ovabumin (50 kDa), car-
bonic anhydrase (36 kDa), ␤-lactolobulin (28 kDa), lysozyme (21 kDa). Lane 2, crude
fermentation broth. Lane 3, purified enzyme.
Fig. 4. Biotransformation of zingiberen newsaponin and deltonin by purified enzyme. S1, zingiberensis newsaponin. S2, deltonin. S3, prosapogenin A of dioscin. P, biotrans-
formed product. The converted products were ascertained by thin-layer chromatography (TLC) on silica gel GF254 plate using CHCl3/CH3OH/H2O = 65:35:10 as developing
solvent. Visualization was performed by 10% H2SO4–EtOH spray reagent, followed by heating.

H. Huang et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 1–7
5
Table 1
Alignment of amino acid sequences of steroidal saponin-␤-glucosidase from A. flavus by ESI-Q-TOF.
Observed
Mr(calc)
Mr(expt)
Delta
Start
End
Peptide
5
5
5
6
7
7
8
9
9
15.3100
47.8300
72.3100
78.3100
52.3200
61.8600
98.4700
45.9500
77.9900
1208.6230
1093.6859
1142.6259
1354.6306
1502.6725
1521.7464
1794.9662
1889.9272
1954.0021
2143.9698
1208.6054
1093.6454
1142.6054
1354.6054
1502.6254
1521.7054
1794.9254
1889.8854
1953.9654
2143.9254
−0.0175
−0.0404
−0.0241
−0.0252
−0.0470
−0.0409
−0.0407
−0.0417
−0.0366
−0.0444
396
850
179
351
191
476
84
747
137
685
405
859
190
361
201
489
99
765
157
703
IGAQSTVLLK
KLPLQASLPK
GIQDAGVIATAK
YTPPNFSSWTR
HYIMNEQEHFR
GNVFAVTDSWALDK
LNIPSLCLQDSPLGIR
LPASGGAGGNPGLYEDLFR
GIDVQLGPAAGPLGAHPDGGR
NFGEIGDASEYVYPEGLER
1
072.9700
Table 2
Alignment of amino acid sequences of steroidal saponin-␤-glucosidase. Matched peptides are boxed in gray.
1
1
MKLGWIEVAA
IVSQMTLTEK
SDYNSAFPAG
AHPDGGRNWE
RQQPEAAGYG
QINNSYGCEN
PGDVTFDSGT
YTPPNFSSWT
TVLLKNKGAL
GSGTANFPYL
LVFVNSDSGE
SVGPVLIDEW
WGKTRESYGS
YGLSYTTFEL
LAAASVVSAK
VNLTTGTGWQ
VNVAATWDKT
GFSPDPALTG
FNVSDSLSSN
SETLNKLLKA
SFWGANLTVG
RDEYGFAHNH
PLSRKEKLVA
VTPEQAIQNE
GYLSVDGNEG
YDHPNVTGIL
PLVKDANNGN
SDLHVQPLNA
WINSTDLKAS
EDLFRVSVKV
EAVWTTTLTR
Q
DDLAYSPPFY
LERCVGQTGS
LAYLRGQAMG
VLFAETIKGI
PSPWADGQGE
VPRLNIPSLC
WAEVYKRAVD
LQDSPLGIRF
QLGPAAGPLG
HYIMNEQEHF
GVGAVMCSYN
AALAGLDMSM
AYYKVGRDTK
DLIRRIGAQS
CDNGTLAMAW
AAAARQASVS
NCNNTVVIIH
VNPGAKSPFT
FNETPIYEFG
5
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
01
51
01
51
01
51
01
51
01
51
01
51
01
51
01
51
EEFSDKGIDV
QDAGVIATAK
LWPFADAVRA
DWTAHHSGVG
VDDMAVRIMA
EFVDVQRDHA
WGANGCDDRG
VTDSWALDKI
GDNVVKTAAN
NSIADVLYGR
VFIDYRHFDK
EAAKNFGEIG
SKYIPEGATD
VPQLYVSLGG
QDWTVTPYPK
VDDKTMHELY
ELGFQGFVMS
VLNGTIPQWR
VSEGAYERVN
LLGEDAGSNS
VLQGRGNVFA
DRNNITLWKN
WAGLPGQESG
GAPQSDFTQG
SRYTPTSGMT
SDDSNYGWED
KNTGNVAGDE
RDLANWDVSA
DASEYVYPEG
GSAQPRLPAS
PNEPKVVLRK
TIYVGNSSRK
LERIHEFIYP
GGAGGNPGLY
FERIHLAPSQ
LPLQASLPKA
Up till now, a few glycosidases with the activity of hydrolyz-
ing dioscin were reported, such as dioscin-␣-l-rhamnosidase from
pig liver [8], dioscin–glycosidase from Absidia sp.d38 stain [9],
and a ␤-glucosidase from A. fumigates (AfG) [12]. Dioscin-␣-
l-rhamnosidase from pig liver hydrolyzes terminal 1,2 linked
rhamnosyl and 1,4 linked rhamnosyl residue of dioscin to trillin as
final product. Dioscin-glycosidase capable of hydrolyzing dioscin to
diosgenin from Absidia sp.d38 was purified. Very recently, a novel
AfG was reported to produce diosgenin from various spirostano-
sides. Interestingly, the new steroidal saponin-␤-glucosidase
described in our study, which was different from the ␤-glucosidases
in the other reports, showed high specificity on the glucose group
of steroidal saponins.
Fig. 6. The optimal temperature on steroidal saponin-␤-glucosidase (0.2
M
Na2HPO4–citric acid buffer pH 5.0).
Fig. 8. Time course of conversion rate ofsteroidal saponin-␤-glucosidase. Gen-
eral properties of purified enzyme was investigated using zingiberen newsaponin
Fig. 7. The thermostability on steroidal saponin-␤-glucosidase (0.2
M
Na2HPO4–citric acid buffer pH 5.0). The steroidal saponin-␤-glucosidase was
(
5
3.0 mg/mL). The purified enzyme was incubated with zingiberen newsaponin at
◦
◦
preincubation at the optimum pH at different temperatures from 30 C to 70 C for
h, and then co-incubated with substrate solution for 24 h.
◦
0
C, 0.2 M Na2HPO4–citric acid buffer pH 5.0, then determined by measuring the
1
increase of diosgenin by HPLC.

6
H. Huang et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 1–7
Fig. 9. Biotransformation of steroidal saponins by steroidal saponin-␤-glucosidase. (a) Biotransformation of zingiberen newsaponin and deltonin. (b) Biotransformation
of prosapogenin A of dioscin to trillin. (c) Biotransformation of trillin to diosgenin. (1) Zingiberen newsaponin and deltonin. (2) Prosapogenin A of dioscin. (3) Trillin. (4)
Diosgenin. P1, transformed product of zingiberen newsaponin and deltonin. P2, transformed product of prosapogenin A of dioscin. P3, transformed product of trillin.
3
.4. The optimal pH, temerature and thermostability of the
The time course of steroidal saponin-␤-glucosidase was
also investigated by measuring the conversion rate at differ-
ent times under optimal conditions. Changes in the relative
conversion rate was summarized in Fig. 8. The conversion
achieved the highest at 96 h then decreased gradually after
96 h.
steroidal saponin-ˇ-glucosidase
The optimum pH of the steroidal saponin-␤-glucosidase was 5.0
as shown in Fig. 5. The range of pH of this enzyme activity was
narrow. The enzyme activity only retained lower than half of the
maximum activity at pH 6.0, and nearly decreased to 0 at pH 3.0.
For the temperature profile (Fig. 6), the optimal temperature of
the enzyme was 50 C, and the activity decreased significantly at
temperatures higher or lower than 50 C. The steroidal saponin-
␤
3.5. Biotransformation of steroidal saponins to diosgenin step by
step and pathways analysis
◦
◦
◦
-glucosidase was stable at 30–40 C, while activity decreased
In our laboratory, it has been found that the steroidal saponin-␣-
1,2-rhamnosidase from C. lunata [10,11] can hydrolyze the terminal
␣-1,2-linked rhamnosyl of dioscin, and only produces secondary
◦
significantly when temperature higher than 60 C, as shown in
Fig. 7.
Fig. 10. Main pathways for biotransformation of zingiberen newsaponin to diosgenin by two key enzymes.

H. Huang et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 1–7
7
saponins. And prosapogenin A of dioscin can be hydrolized to trillin
by the steroidal saponin-␣-1,2-rhamnosidase.
The present study elucidates firstly the key enzymes of every
step in bioconversion of two main steroidal saponins from DZW,
namely zingiberen newsaponin and deltonin, to diosgenin. This
makes it possible to apply gene engineering to express the key
enzymes to a higher level, so that the cost of biotransformation can
be reduced, facilitating future industrial production of diosgenin by
biotransformation.
Therefore, the steroidal saponin-␣-1,2-rhamnosidase and the
␤
-glucosidase from A. flavus were used for biotransformation of
zingiberen newsaponin and deltonin to diosgenin step by step.
Both zingiberen newsaponin and deltonin can be converted to a
compound with lower polarity by steroidal saponin-␤-glucosidase
(Fig. 9a). The product was purified and identified to be prosa-
pogenin A of dioscin. In the second step, prosapogenin A of dioscin
was used as the substrate. From Fig. 9b, more than half of pros-
apogenin A of dioscin was transformed to trillin after 72 h by
incubated with steroidal saponin-␣-1,2-rhamnosidase. The ter-
minal ␤-glucoside residue of trillin was finally hydrolyzed to
diosgenin by steroidal saponin-␤-gluosidase as shown in Fig. 9c.
Based on the results above, the possible and main pathways
for the conversion of zingiberen newsaponin to diosgenin by two
enzymes, steroidal saponin-␤-gluosidase and steroidal saponin-␣-
Acknowledgements
We are grateful to Shuo Cheng of the Beijing Proteome Research
Center for ESI-Q-TOF measurements. This work was financially
supported by the Major Program of Municipal Natural Science
Foundation of Beijing (No. 7090001), the National Natural Science
Foundation of China (No. 81202417), and National Infrastructure of
Microbial Resource (No. NIMR-2012-3).
1
,2-rhamnosidase, were deduced in Fig. 10.
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(
data not shown).