ALDEHYDE DEHYDROGENASE FROM PSEUDOMONAS NITROREDUCENS
3
af final concentration) and carried out at 30°C for
30 min. The rate of decrease in the absorbance at
600 nm was followed against a blank in which
20 mM Tris-HCl buffer (pH 8.0) was added in place
of enzyme. One unit of BMAL-CHO dehydrogenase
activity was defined as 1 μmol DCIP reduced
per minute per liter of enzyme solution. Protein con-
centration was quantified using Pierce Coomassie
Protein Assay kit (Thermo Fisher Scientific,
Waltham, MA, USA) with bovine serum albumin
standard.
100 mL of 20 mM Tris-HCl buffer (pH 8.0). The active
fractions were combined and concentrated, and the
buffer was exchanged to 20 mM Tris-HCl buffer (pH
8.0) with 50 mM NaCl by ultrafiltration (Amicon
Ultra; Merck Millipore, Burlington, MA, USA). The
enzyme solution was applied to a Superdex 200 10/300
GL (1.0 × 30 cm) (GE Healthcare) previously equili-
brated with 20 mM Tris-HCl buffer (pH 8.0) contain-
ing 50 mM NaCl and eluted with the same buffer. The
active fractions were used for characterization. The
purity of the proteins in the active fractions was
checked by SDS-PAGE using a 12.5% polyacrylamide
gel stained with Coomassie Brilliant Blue R-250. The
protein bands were cut from the gel and transferred to
a PVDF membrane. The N-terminal amino acid
sequences of the proteins were determined by auto-
mated Edman degradation with a PSQQ-30 Protein
Sequencer (Shimadzu).
Purification of a BMAL-CHO-oxidizing enzyme
from Pseudomonas nitroreducens SB32154
For the purification of a BMAL-CHO-oxidizing
enzyme, P. nitroreducens SB32154 was cultivated in
1 L LB medium containing 10 mL ethanol. Cultivation
was carried out at 30°C for 24 h with agitation at
80 rpm on a rotary shaker. Bacterial cells were har-
vested by centrifugation at 5,500 × g for 15 min at 4°C.
The resulting cell pellets were resuspended in 20 mM
Tris-HCl buffer (pH 8.0) containing 2 mM DTT and
disrupted with a probe sonicator (Branson Sonifier
450; Emerson, St. Louis, MO, USA). The parameter
was set at output of 6 at 20% duty cycle in 10 s pulse
mode for 30 min. The cell debris was removed by
centrifugation at 8,000 × g for 30 min at 4°C. The
0.22 µm filtrated cell-free supernatant was designated
as crude BMAL-CHO dehydrogenase. All purification
procedures were performed at 4°C using an ÄKTA
FPLC system (GE Healthcare, Little Chalfont, UK).
The crude BMAL-CHO dehydrogenase was applied
to a HiPrep DEAE 16/10 FF column (1.6 × 10 cm) (GE
Healthcare) previously equilibrated with 20 mM Tris-
HCl buffer (pH 8.0), which was used as a standard
buffer for the following purification steps. After the
column had been washed with 40 mL of this buffer, the
enzyme was eluted with a linear gradient of NaCl
(0–0.4 M) in 100 mL of 20 mM Tirs-HCl buffer (pH
8.0). The active fractions were combined and dialyzed
against 5 L of 20 mM Tris-HCl buffer (pH 8.0) for 24 h
using Slide-A-Lyze 10 kDa molecular weight cutoff
cassettes (Thermo Fisher Scientific). The enzyme solu-
tion was applied to Mono Q 5/50 GL column
(0.5 × 5 cm) (GE Healthcare) previously equilibrated
with 20 mM Tris-HCl buffer (pH 8.0). After the col-
umn had been washed with 2 mL of 20 mM Tris-HCl
buffer (pH 8.0), the enzyme was eluted with a linear
gradient of NaCl (0–0.3 M) in 20 mL of 20 mM Tris-
HCl buffer (pH 8.0). The active fractions were com-
bined, added with the same volume of 1.6 M (NH4)2
SO4, and then applied to HiTrap Butyl FF 5 mL col-
umn (1.6 × 2.5 cm) (GE Healthcare) previously equi-
librated with 20 mM Tris-HCl buffer (pH 8.0)
containing 0.8 M (NH4)2SO4. The enzyme was eluted
with a linear gradient of (NH4)2SO4 (0.8–0 M) in
Characterization of BMAL-CHO dehydrogenase
The effect of different electron acceptors was evaluated
with the purified enzyme from P. nitroreducens
SB32154 in the standard reaction mixture with each
electron acceptor instead of DCIP and PMS. The reac-
tion was carried out at 30°C for 30 min. The enzyme
activity was evaluated by the production of BMAL-
COOH using liquid chromatography-mass spectro-
metry (LC-MS).
To determine the substrate specificity, the assay
conditions were the same as mentioned above, except
that different aldehydes were used as the substrate
instead of BMAL-CHO. Separate blanks with indivi-
dual substrates of aldehyde were also prepared. The
kinetic parameters, such as Michaelis-Menten con-
stant (Km) and maximum reaction velocity (Vmax
)
values against aldehyde substrates, were calculated
using XLfit (IDBS, Guilford, UK).
Cloning and expression of recombinant
BMAL-CHO dehydrogenase in Escherichia coli
Whole-genome sequencing (WGS) of P. nitroreducens
SB32154 was conducted by Genaris, Inc. (Yokohama,
Japan). Primers were designed to amplify the BMAL-
CHO dehydrogenase sequence from P. nitroreducens
SB32154
genomic
DNA
(i.e.
Fw:
5ʹ-
TCACATATGATTACCGTGAACCTGAACGGCAAG-
GAC-3ʹ and Rev: 5ʹ-ATACTCGAGTCAGGCC
TGCAACTGATTGCCGATC-3ʹ). The polymerase chain
reaction-amplified product was ligated into expression
vector pET17b (Novagen, Madison, WI, USA) using
NdeI and XhoI restriction enzyme sites. The resulting
plasmid was purified and then used to transform E. coli
BL21 STAR (DE3) (Thermo Fisher Scientific). The trans-
formed cells were cultured in Luria-Bertani (LB) medium
at 37°C for 2 h with shaking at 150 rpm, and then
Hibi, Makoto
