J. Yang et al. / Process Biochemistry 47 (2012) 720–724
721
2. Materials and methods
2.1. Reagents
4-Nitro-1,8-naphthalic anhydride (95% purity), reduced NADH and NADPH,
phenazine methosulfate and nitro-blue tetrazolium (NBT) were purchased from
Sigma–Aldrich (Shanghai, China). FMN, FAD, and riboflavin were purchased from
J & K. 4-Amino-1,8-naphthalic anhydride was a gift of Professor Xuhong Qian, East
China University of Science and Technology. Other analytical grade solvents were
purchased from Fluka.
2.2. Bacterial strains and culture conditions
The strain S. mirabilis DUT001 was obtained from a preserved sample from the
China General Microbiological Culture Collection Center (CGMCC), Beijing, China
(CGMCC number is 2517). It was maintained in a liquid medium containing (g L−1):
soluble starch 10.0, glucose 20.0, bean flour 25.0, and beef extract 1.0, K2HPO4 0.05,
NaCl 2.0 and yeast extract 4.0.
2.3. Nitroreductase assay
Nitroreductase activity was determined with an assay mixture containing
50 g of protein, 0.1 mM of 4-nitro-1,8-naphthalic anhydride solution dissolved
in dimethyl sulfoxide (DMSO), and 0.2 mM of NADH, in 20 mM Tris buffer (pH
8.0), to a final volume of 200 L. 4-Nitro-1,8-naphthalic anhydride and its reduced
products were visible by normal phase thin layer chromatography (TLC) and sup-
ported by HPLC analysis[17]. After ethyl acetate extraction, the biotransformation
products were separated on a reversed-phase C18 column (Kromasil C18, 10 M,
4.6 mm × 250 mm) with a linear gradient of 30–100% methanol as a solvent (con-
taining 1 g L−1 citric acid), at a flow rate of 1 mL min−1. The detection wavelength
was 254 nm and the column temperature was kept at 30 ◦C. Retention times were
21 min for 4-amino-1,8-naphthalic anhydride and 29 min for 4-nitro-1,8-naphthalic
anhydride. One unit of enzyme activity was defined as the amount of activity needed
to catalyze 1 mol of amine product per min.
Fig. 1. SDS-PAGE of the purified nitroreductase from Streptomyces mirabilis DUT001.
Lane M: molecular mass of standards. Lane 1: the purified nitroreductase.
analyzed using TLC and HPLC. The effect of temperature on nitroreductase activ-
ity was determined. Purified nitroreductase was incubated in a water bath with a
temperature range from 20 to 60 ◦C. After 10 min incubation, 20 g of protein was
added to an assay mixture adjusted to the same temperature, and the products were
analyzed using HPLC. The N-terminal sequence of purified nitroreductase was ana-
lyzed by the Edman degradation method using an Automated Protein Sequencer,
model ABI Procise 492 cLC. The effects of metal ions on nitroreductase activity
were determined after adding appropriate amounts of corresponding salts. Effects
of surfactants, solvents, inhibitors and other chemicals were determined by adding
various concentrations of these compounds to the reaction vials before incubation.
Otherwise, all assays were performed as described above.
2.4. Purification of nitroreductase from S. mirabilis DUT001
Cells of S. mirabilis DUT001 grown in nutrient broth medium for four days were
harvested by centrifugation, at 4 ◦C, and 7500 rpm. Pellets were washed twice in
20 mM Tris buffer (pH 7.5). 10 g wet cell paste was ground with 0.5 mm diameter
glass beads to a fine powder under liquid nitrogen for 0.5 h, and was then suspended
in 50 mM Tris buffer (pH 7.5). The supernatant fluid was centrifuged at 4 ◦C and
12,000 rpm, and the pellet was discarded. The supernatant was fractionated with
70% saturated solid ammonium sulfate, and the precipitate obtained by centrifu-
gation at 12,000 rpm for 30 min was suspended in 20 mM Tris–HCl buffer (pH 8.0).
The supernatant was then collected and desalted on a HiPrep 26/10 desalting col-
umn (50 mL; GE Healthcare, Uppsala, Sweden) with the same buffer. The resulting
liquid was subjected to column chromatography using an ÄKTA purifier 100 (GE
Healthcare Biosciences, Sweden). The sample was loaded onto a Sepharose Q anion
exchange column (12 mL; GE Healthcare) pre-equilibrated with 20 mM Tris–HCl
buffer (pH 8.0) at a flow rate of 1 mL min−1, and eluted with an increasing linear
gradient of 0–0.2 M NaCl buffer (20 mM Tris–HCl, 1.0 M NaCl, pH 8.0) at a flow rate
of 2 mL min−1. Fractions were analyzed for nitroreductase activity and active frac-
tions were pooled and ultrafiltered to a volume of 1.0 mL. Each sample was loaded
onto a Superdex 200 10/30 GL column (24 mL; GE Healthcare), pre-equilibrated with
20 mM Tris–HCl buffer (100 mM NaCl, pH 8.0). Pooled fractions of the active peaks
were eluted with the same buffer at a flow rate of 1.0 mL min−1. Aliquots of purified
nitroreductase were stored at −20 ◦C.
2.6. Enzymatic transformation and kinetic parameters of purified nitroreductase
The kinetic parameters for NADH as an electron donor were determined by
changing its concentration under standard assay conditions using 0.05–1 mmol/L
tron acceptors. Kinetic parameters for 4-nitro-1,8-naphthalic anhydride and
4-nitrophthalimide were determined with concentrations of NADH from 0.01
to 0.05 mM. In addition, the inhibition profiles of four inhibitors (menadione,
dicoumarol, sodium benzoate, and antimycin A) were investigated. The inhibition
constants (Kis) were determined using a Dixon plot [18]. Reaction mixtures were
incubated at 37 ◦C, and reactions were stopped by extraction of the mixture with
ethyl acetate. HPLC was used to quantitate nitroaromatic substrates and transfor-
mation products.
2.7. Substrate specificity
2.5. Characterization of purified nitroreductase
The substrate specificity of purified recombinant nitroreductase was deter-
mined in the presence of different nitroaromatic compounds, including 4-nitro-
1,8-naphthalic anhydride, 3-nitro-1,8-naphthalic anhydride, 1,3-dinitrobenzene,
1,4-dinitrobenzene, 3-nitrophthalimide, 2-nitrotoluene and nitrobenzene (in a final
concentration of 1 mM), incubated at 30 ◦C for 30 min. The relative activity of nitrore-
duction was calculated compared with 4-nitro-1,8-naphthalic anhydride (4-NNA).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed to determine the relative molecular mass of the denatured nitroreduc-
tase with molecular weight standards. The molecular mass of the native enzyme was
estimated by gel filtration on a Superdex 200 column (GE Healthcare) calibrated with
low-molecular-weight calibration Kits (GE Healthcare).
UV–visible absorption spectrum of the purified nitroreductase at 0.5 mM was
measured in 20 mM Tris–Cl buffer, pH 7.4. For comparison, the absorption spectra
of free FAD, FMN and riboflavin at 0.1 mM were also measured.
3. Results and discussion
Nitro-blue tetrazolium (NBT) NAD(P)H depletion assays[12] were used to anal-
ysis the cofactor consumption. Reactions were performed in 20 mM Tris–Cl (pH 8.0),
4% DMSO at room temperature for 3 or 30 min, initiated by the addition of 0.05 mM
NADH or NADPH. Substrates were 4-nitro-1,8-naphthalic or 4-nitrophthalimide at
the concentration ranges from 0 to 0.5 mM. Reactions were halted by addition of
50 L NBT solution. To quantify the amount of NAD(P)H remaining, the initial rates
of blue-colored formazan production were monitored at 590 nm.
The effect of pH on nitroreductase activity was determined using 20 mM phos-
phate buffer (pH 5.5–7.0), and 20 mM Tris–HCl buffer (pH 7.0–8.5). The reaction
mixture contained 20 g protein, 0.1 mM 4-nitro-1,8-naphthalic anhydride, 0.2 mM
NAD(P)H, and 20 mM of buffer in a final volume of 0.2 mL at each pH. Products were
3.1. Biochemical characterization
from a culture of S. mirabilis DUT001 is outlined in Table 1. This
enzyme was purified about 33 fold with an overall yield of 32%. SDS-
polyacrylamide gel electrophoresis of the purified nitroreductase
from S. mirabilis DUT001 gave a single band with molecular weight
of 34 kDa (Fig. 1). However, the molecular weight of the enzyme by