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M. Nojiri et al. / Journal of Molecular Catalysis B: Enzymatic 109 (2014) 136–142
derivatives. The conversion degree analysis of NPD and NPA deriva-
tives was performed by reverse-phase HPLC with a Shimadzu LC-VP
system equipped with a YMC-A303 column (4.6 mm × 250 mm).
HPLC was conducted using acetonitrile:water (3:7 by vol., pH 2.5,
adjusted with phosphoric acid) as the mobile phase, a flow rate
of 1.0 mL/min, a column temperature of 35 ◦C, and UV detec-
tion at 210 nm. The determination of the optical purity of NPD
derivatives was performed by reverse-phase HPLC using a Shi-
madzu LC-VP system equipped with a CHIRALPACK AD-RH column
(4.6 mm × 150 mm). HPLC was conducted using acetonitrile:water
(3:7 by vol., pH 2.5, adjusted with phosphoric acid) as the mobile
phase, a flow rate of 0.5 mL/min, a column temperature of 30 ◦C,
and UV detection at 210 nm.
2.7. Determination of enzyme molecular mass
The molecular mass of the native enzyme was estimated by
column chromatography using a Superdex 200 HR 10/30 col-
umn (24 mL; GE Healthcare Bio-Sciences) and standard molecular
markers in 50 mM potassium phosphate buffer (pH 7.0) contain-
ing 150 mM sodium chloride. The molecular mass of the subunit
was estimated by SDS-PAGE (10%) with SDS-PAGE markers as the
standard.
2.8. Partial amino acid sequence
CsAM was separated from other peptides by HPLC on a YMC-
Pack PROTEIN-RP column (4.6 × 250 mm; YMC) equilibrated with
0.1% trifluoroacetic acid and eluted with a linear acetonitrile gradi-
ent (10%–70%) at a flow rate of 1.0 mL/min. The HPLC-purified CsAM
was digested with lysyl endopeptidase (Wako Pure Chemicals) in
4 M urea and 50 mM Tris–HCl buffer (pH 9.0) for 24 h at 30 ◦C.
Then, the peptides were separated by HPLC as described above.
The sequence was analyzed with a model 477A gas–liquid-phase
protein sequencer (Applied Biosystems, Carlsbad, CA, USA).
2.6. Purification of the BNPD-hydrolyzing enzyme CsAM from
Cupriavidus sp. KNK-J915
Cupriavidus sp. KNK-J915 was subcultured at 30 ◦C for 24 h in a
test tube containing 8 mL of CM medium. The subculture (1 mL) was
then inoculated into a 500-mL Sakaguchi flask containing 120 mL
of medium P. After a 3-day incubation at 35 ◦C with reciprocal shak-
ing, Cupriavidus sp. KNK-J915 cells were collected by centrifugation
from 4.2 L of the cultured broth (from Sakaguchi flask × 36), washed
with 100 mL of 100 mM potassium phosphate (pH 7.0), and sus-
pended in 200 mL of 100 mM potassium phosphate (pH 7.0). All
purification procedures were performed at 4 ◦C. After ultrasonic
disruption of the cells, using an ultrasonic homogenizer UH-600 S
(SMT Co., Ltd., Tokyo, Japan) for 10 min (pulse 50%, output 10,
1 min × 10), 151 mL of cell-free extract were obtained by centrifu-
gation. Ammonium sulfate was added to the cell-free extract to
reach a saturation concentration of 20%, and the mixture was stirred
for 0.5 h. The precipitate was discarded after centrifugation. In addi-
tion, ammonium sulfate was added to the supernatant to reach a
saturation concentration of 40%. After being stirred for 0.5 h, the
mixture was centrifuged to obtain the formed precipitates, and the
precipitates were suspended in 40 mL of 100 mM potassium phos-
phate (pH 7.0) and dialyzed with 10 mM potassium phosphate (pH
8.0). The dialyzed enzyme solution (67 mL) was applied to a 400-mL
Toyopearl DEAE-650 M column (Tosoh, Tokyo, Japan) equilibrated
with 10 mM potassium phosphate (pH 8.0). The enzyme was eluted
with a 0–0.3 M sodium chloride linear gradient. In total, 200 mL of
enzyme solution of the active fractions were collected and dialyzed
with 10 mM potassium phosphate (pH 7.0). Ammonium sulfate
was added to the dialyzed enzyme solution to give a final con-
centration of 0.8 M. The enzyme solution (190 mL) was applied
to a 75-mL Toyopearl Phenyl-650 M column (Tosoh) equilibrated
with 10 mM potassium phosphate (pH 7.0) containing 0.8 M ammo-
nium sulfate. The enzyme was eluted with a 0–0.8 M ammonium
sulfate linear gradient. In total, 30 mL of enzyme solution of the
active fractions were collected and dialyzed with 10 mM potassium
phosphate (pH 7.0). Ammonium sulfate was added to the dialyzed
enzyme solution to give a final concentration of 0.8 M. The enzyme
solution (26 mL) was applied to a 25-mL Toyopearl Butyl-650S col-
umn (Tosoh) equilibrated with 10 mM potassium phosphate (pH
7.0) containing 0.8 M ammonium sulfate. The enzyme was eluted
with a 0–0.8 M ammonium sulfate linear gradient. Ten milliliters
of the enzyme solution of the active fractions were collected and
dialyzed with 10 mM potassium phosphate (pH 8.0). The dialyzed
enzyme solution (9.5 mL) was applied to a 6-mL Resource Q column
(GE Healthcare Bio-Sciences) equilibrated with 10 mM potassium
phosphate (pH 8.0). The enzyme was eluted with a 0–0.5 M sodium
chloride linear gradient. In total, 1.9 mL of the enzyme solution of
the active fractions were collected. The active fraction was used as
the purified enzyme.
2.9. General recombinant DNA techniques
General DNA manipulations were conducted as described by
Sambrook and Russell [12]. Genomic DNA from Cupriavidus sp.
KNK-J915 was prepared using a GNOME DNA isolation kit (MP
Biomedicals, Santa Ana, CA, USA). Plasmid DNA was purified from
E. coli using a QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany).
DNA fragments were recovered from agarose gels using a QIAquick
Gel Extraction Kit (Qiagen). Restriction enzymes, a DNA Ligation Kit,
T4 DNA ligase, and Ex Taq DNA polymerases for polymerase chain
reaction (PCR) were purchased from Takara Bio (Shiga, Japan).
2.10. PCR for the core region of the CsAM gene
PCR amplification was performed in
(50 L) containing 100 ng of chromosomal DNA as
a
reaction mixture
tem-
a
plate, both primers (40 pmol each), four dNTPs (final con-
centration, 10 nmol each), and 2.5 U of Ex Taq DNA poly-
merase in the buffer for the polymerase. Two oligonucleotide
primers, CsAM-F (5ꢀ-GAYATHCARACNTTRCARAC-3ꢀ) and CsAM-C
(5ꢀ-CCNGCDATRTTRAANAGRTC-3ꢀ) were synthesized and used. The
reaction was performed for 30 cycles (1 min at 95 ◦C, 1 min at 50 ◦C,
and 0.5 min at 72 ◦C) using a program temperature control sys-
tem PC-701 (ASTEC, Fukuoka, Japan). The DNA fragment amplified
by PCR was purified and ligated with a pT7Blue T-vector (Merck,
Darmstadt, Germany) using a DNA Ligation Kit.
2.11. Inverse PCR for DNA sequences flanking the core region
digested with PstI for 18 h at 37 ◦C. The digested DNA was
circularized with T4 DNA ligase. Two oligonucleotide primers,
CsAM-iF (5ꢀ-TGGCAGGCCTGCCGGTTTCGGTCA-3ꢀ) and CsAM-iR (5ꢀ-
GGAGACCGCGCCATCGTGAAGTCT-3ꢀ), were synthesized. Amplifi-
cation by inverse PCR [13] was performed in reaction mixtures
(50 L) containing 200 ng of circularized DNA obtained as previ-
ously described, both primers (50 pmol each), four dNTPs (final
concentration, 10 nmol each), and 2.5 U of Ex Taq DNA polymerase
in the buffer for the polymerase. The reaction was performed for
30 cycles (1 min at 97 ◦C, 1 min at 60 ◦C, and 5 min at 72 ◦C). The
amplified DNA fragment was ligated with a pT7Blue T-vector.