G Model
PRBI-10323; No. of Pages7
ARTICLE IN PRESS
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M.-M. Zheng et al. / Process Biochemistry xxx (2015) xxx–xxx
The cells were cultivated in LB medium containing 50 g mL−1
kanamycin at 37 ◦C. When the OD600 reached 0.6–0.8, isopropyl
-d-1-thiogalactopyranoside (IPTG) was added to a final concen-
tration of 0.2 mM. After cultivation at 16 ◦C for an extra 24 h, the
cells were centrifuged at 8800 × g for 10 min and washed with nor-
mal saline. The cells were resuspended in buffer A (20 mM sodium
phosphate buffer pH 7.4, 500 mM NaCl and 10 mM imidazole), and
broken by sonication with an ultrasonic oscillator (JY92-II, Scientz
Biotech Co.). The cell lysate was centrifuged at 12.000 × g for 20 min,
and then the supernatant was filtered and loaded onto a Ni-NTA
column (5 mL, GE Healthcare Co.) equilibrated with buffer A. After
prewashing with buffer A, the column was eluted with a linear gra-
dient from 10 to 500 mM of imidazole in buffer A at 5 mL min−1. The
purity of fractions was assessed by sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE). Fractions containing
7-hydroxysteroid dehydrogenase (7-HSDH) activity were col-
lected and dialyzed extensively against sodium phosphate buffer
to remove the high concentrations of imidazole and salt.
tion of CDCA to UDCA. Genes encoding 7␣-HSDH have been cloned
from Eubacterium sp. [17], Escherichia coli [18], Clostridium sordellii
highest activity for converting CDCA into UDCA [22]. In contrast,
much less information is available for 7-HSDH. So far, only three
genes encoding 7-HSDHs have been cloned from C. absonum [21],
Collinsella aerofaciens [23] and Ruminococcus gnavus [24]. Therefore,
it is necessary to find more and better 7-HSDHs for efficient syn-
thesis of UDCA. Herein, we report a new and highly active 7-HSDH
whose gene was successfully cloned from Ruminococcus torques
fied and its enzymatic properties were characterized.
Meanwhile, we describe the biotransformation of CDCA to UDCA
using 7␣-HSDH and 7-HSDH via sequential two-step reactions in
one pot (Fig. 2). In the oxidative step, CDCA was oxidized to 7-oxo-
lithocholic acid (7-oxo-LCA) in the presence of 7␣-HSDH, coupled
by the regeneration of cofactor (NAD+) with lactate dehydrogenase
(LDH) and pyruvate. Similarly, 7-HSDH catalyzed the reduction of
7-oxo-LCA to UDCA with glucose dehydrogenase (GDH) and glucose
for the regeneration of cofactor (NADPH) in the reductive step. 7␣-
HSDH and LDH were inactivated by heating prior to the second
step to avoid the reversible reaction in order to increase the yield
of UDCA.
2.3. Protein assays
The enzyme activity was determined spectrophotometrically at
340 nm (ε = 6.22 mM−1 cm−1) and 30 ◦C by measuring the oxidation
of NAD(P)H or reduction of NAD(P)+.
The standard assay mixtures (1 mL) were: (a) For the NAD(P)H-
dependent 7␣-HSDH assay: 0.1 mM NAD(P)+, 1 mM CDCA in 0.1 M
phosphate buffer, pH 8.0; (b) For the 7-HSDHRt assay: 0.1 mM
NADP(H), 1 mM 7-oxo-LCA (or UDCA) in 0.1 M phosphate buffer,
pH 6.5 (or 8.0); (c) For the LDH assay: 0.1 mM NADH, 1 mM sodium
pyruvate in 0.1 M phosphate buffer, pH 8.0; (d) For the GDH assay:
0.1 mM NADP+, 1 mM glucose in 0.1 M phosphate buffer, pH 6.5, and
an appropriate amount of enzyme. One unit of activity is defined
as the amount of enzyme catalyzing the reduction (or oxidization)
of 1 mol NAD(P)(H) per min under the assay conditions used.
2. Materials and methods
2.1. Materials
The microbial strains used for genome mining were obtained
from the American Type Culture Collection (ATCC), German
Collection of Microorganisms and Cell Cultures (DSMZ), and
E. coli DH 5␣ and E. coli BL21 (DE3) were used as the cloning
(7␣-HSDH, EC 1.1.1.159) from E. coli [18], NADPH-dependent
7␣-hydroxysteroid dehydrogenase from C. absonum DSM 599
[21], lactate dehydrogenase (LDH, EC 1.1.1.27) from Lactobacillus
delbrueckii subsp. bulgaricus DSM 20081 [25], and glucose dehy-
drogenase (GDH, EC 1.1.1.47) from Bacillus megaterium [26] were
overexpressed in recombinant E. coli BL21(DE3). Chenodeoxy-
cholic acid (CDCA; 3␣,7␣-dihydroxy-5-cholan-24-oic acid) and
ursodeoxycholic acid (UDCA; 3␣,7-dihydroxy-5-cholan-24-oic
acid) were purchased from Shanghai Siyu Chemical Technology
Co., Ltd. (Shanghai, China). 7-Oxo-lithocholic acid (7-oxo-LCA;
3␣-hydroxy-7-oxo-5-cholan-24-oic acid) was purchased from
Mairuier Chemical Technology Co., Ltd. (Shanghai, China). All
the other chemicals used in this work were obtained from
commercial sources and were of reagent grade or better
quality.
2.4. Characterization of purified 7ˇ-HSDHRt
The effect of temperature was determined by assaying the 7-
HSDHRt activity at different temperatures in the range of 20–50 ◦C
in phosphate buffer (100 mM, pH 6.5). The optimum pH was deter-
mined using the standard activity assay at different pHs (5.0–9.0
for the forward reaction, 6.5–10.5 for the reverse reaction). The
thermostability of 7-HSDHRt was investigated by incubating the
purified enzymes (1.0 mg mL−1) in the same phosphate buffer
(100 mM, pH 6.5) at 30 ◦C, 40 ◦C and 45 ◦C, and measuring the
residual activities at different times. The stability of 7-HSDHRt at
different pH was investigated by incubating the purified enzymes
(1.0 mg mL−1) in the phosphate buffer (100 mM, pH 6.5), phosphate
buffer (100 mM, pH 8.0) and Gly-NaOH (100 mM, pH 10.0) at 30 ◦C,
and measuring the residual activities at different times.
The kinetic parameters of the 7-HSDHRt in both direc-
tions were determined by non-linear fitting using Microsoft-Excel
2010. The substrate, 7-oxo-LCA, at 0.008 mM, 0.01 mM, 0.015 mM,
0.02 mM, 0.025 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM and 0.8 mM,
was used for the enzyme activity assay using the standard method.
The concentrations of UDCA tested included 0.01 mM, 0.02 mM,
0.03 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM and 1.0 mM.
2.2. Cloning, expression and purification of 7ˇ-HSDHRt
The genomic DNA of R. torques ATCC 35915 was extracted
and purified using the TIANamp Bacteria DNA Kit from Tiangen
(Shanghai, China). DNA fragments containing the 7-HSDHRt gene
were amplified by polymerase chain reaction (PCR) using primers
with BamH I and Sal I restriction sites. The resulting plasmid was
transformed into E. coli DH5␣ for amplification. The plasmid was
extracted using standard methods and then transformed into E. coli
BL21 (DE3) cells.
2.5. One-step cascade reaction in one-pot transformation of
CDCA to UDCA using 7˛-HSDHCa and 7ˇ-HSDHRt
7␣-HSDHCa from C. absonum DSM 599 is an NADPH-dependent
enzyme. The 5-mL reaction starting from the CDCA consisted of
100 mM phosphate buffer (pH 8.0), 10 mM CDCA, 0.5 mM NADP+,
combined with 0.5 U mL−1, 1.0 U mL−1, or 2.0 U mL−1 7␣-HSDHCa
and 7-HSDHRt respectively. The 5-mL reaction starting from the
Please cite this article in press as: Zheng M-M, et al. Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7-hydroxysteroid