10.1002/adsc.201900849
Advanced Synthesis & Catalysis
Enzyme activity was determined by spectrophotometric
assay at 30°C measuring the oxidation of NAD(P)H or
reduction of NAD(P)+ at 340 nm (ɛ = 6.22 mM−1 cm−1).
The reaction mixture (1 mL) was composed of 970 μL
potassium phosphate buffer (KPi, 100 mM, pH 8.0), 10 μL
of substrate stock solution (200 mM), 10 μL NAD(H) or
NADP(H) solution (100 mM), and 10 μL purified enzyme
with an appropriate concentration. One unit of the enzyme
activity is defined as the amount of enzyme that catalyzes
the reduction (or oxidation) of 1 μmol NAD(P)(H) per
minute under the assay conditions. Kinetic parameters
were determined at different substrate concentrations (0–
10 mM) and cofactor (NAD+ or NADP+) concentrations
(0–1.0 mM). The resultant data were adjusted to a
Michaelis-Menten model using Origin 8.6.
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Preparative Synthesis of 12-Oxo-hydroxysteroids
The biotransformation was conducted at 30°C and 220 rpm
for 24 h in a 100 mL solution containing 150–200 mM
12α-hydroxysteroids, 1.5-fold equivalent pyruvate, 5 g L-1
each of lyophilized E. coli cells harboring Rr12α-HSDH
and LdLDH genes respectively, 0.5 mM NAD+, and
potassium phosphate buffer (KPi, 100 mM, pH 8.0). The
conversion of substrates was determined by RP-HPLC
(Supporting Information). After complete conversion, the
solution was acidified with 1 M HCl till pH 3.0, and the
precipitated product was filtered and washed with 50−100
mL of ethanol. Subsequently, the ethanol is evaporated,
and the powder was dried, and purified by silica gel
column chromatography eluting with a mixture of
dichloromethane and methanol (20/1–10/1) to yield pure
12-oxo-hydroxysteroids. The product was characterized by
NMR spectroscopy and high-resolution mass spectrometry
(HRMS) (Supporting Information).
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Acknowledgements
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This work was financially supported by The National Key
Research and Development Program of China (No.
2018YFC1706200), The National Natural Science Foundation of
China (Nos. 21536004, 21871085, and 31500592), and the
Fundamental Research Funds for the Central Universities (No.
22221818014). We are grateful to Dr. Yu-Cong Zheng and Dr.
Chao Shou, both from East China University of Science and
Technology, for their experimental assistance.
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