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H. China, H. Ogino / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
external factors, including various carboxylic acids as cofactors and
several alcohols as water-miscible organic solvents, on the activity
of the organic solvent-tolerant rBPO-A1.
(Wako Pure Chemical, Industries, Ltd.) as an indicator [22]. The
protein concentration was determined by the Bradfold method
using bovine serum albumin as a standard [23].
2. Materials and methods
2.3. Non-enzymatic and enzymatic brominating activities
2.1. Preparation of purified rBPO-A1
Non-enzymatic oxidative bromination using peracetic acid
(AcOOH), was performed to identify the products. The reaction
mixture was composed of 1.0 M AcOH-NaOH buffer (pH 5.5),
10 mM H2O2, 0.5 M NaBr, 30% (v/v) MeOH, 20 mM substrate
(cyclohexene, indene, or nerol), and 20 mM AcOOH. After the
mixture was stirred at 1 ꢁC for 0.5 h, organic compounds were
extracted thrice with 50 mL of dichloromethane. The organic phase
was washed twice with 100 mL of water, and the extract was then
filtered through a filter paper for dehydration. After removing of
the solvent by evaporation below 10 ꢁC, the residue was purified by
silica gel chromatography with AcOEt/hexane (2/8 and 3/7). The
isolated products were identified by 1H and 13C NMR analyses.
Enzymatic oxidative bromination using rBPO-A1 was performed
to detect of the products. The reaction mixture was composed of
0.3 M propanoic acid-NaOH buffer (pH 5.5), 500 mM NaBr,
21 mM H2O2, 10 mM NaN3, 25% (v/v) MeOH, 1% (v/v) substrates
(cyclohexene, indene, or nerol), and 8 U rBPO-A1. After the reaction
mixture was incubated at 30 ꢁC for 24 h, organic compounds were
extracted thrice with dichloromethane. The organic phase was
washed with water, and the extract was filtered through a filter
paper for dehydration. After removal of the solvent by evaporation
below 10 ꢁC, the crude products were identified by 1H NMR analysis.
E. coli Rosetta™2 (DE3) cell transformed with pET42a(þ)_BPO-
A1-His [14], were cultured in 200 mL of liquid Luria-Bertani (LB)
medium (Miller) and supplemented with chloramphenicol (17 mg/
L) and kanamycin (30 mg/L) at 37 ꢁC. When culture OD660 reached
0.6, bpo-A1-his gene expression was induced by addition of 1.0 mM
isopropyl-b-D-thiogalactopyranoside (IPTG). After incubation at
37 ꢁC for 6 h, the transformed cells were collected by centrifugation
and re-suspended in 10 mL of 0.2 M Tris-H2SO4 buffer (pH 8.3). The
suspended cells were disrupted by ultrasonic disintegration using
an ultrasonic disruptor UD-200 (Tomy Seiko Co., Ltd., Tokyo, Japan)
at 80 W for 10 min, intermittently, in an ice bath. Cell debris was
removed by centrifugation at 27,700 ꢂ g at 4 ꢁC for 10 min, and the
supernatant was obtained.
Crystalline ammonium sulfate was slowly dissolved in the su-
pernatant by stirring at 4 ꢁC for 30 min to be 30% (w/v) final satu-
ration. After the precipitate was removed by centrifugation at
17,700 ꢂ g at 4 ꢁC for 10 min, additional crystalline ammonium
sulfate was also slowly dissolved in the resulting supernatant by
stirring at 4 ꢁC for 30 min to be 50% (w/v) final saturation. The
resulting precipitate was collected by centrifugation at 17,700 ꢂ g at
4 ꢁC for 10 min and re-dissolved in 1 mL of 0.2 M Tris-H2SO4 buffer
(pH 8.3).
3. Results and discussion
The rBPO-A1 solution obtained by ammonium sulfate fraction-
ation, was diluted to 5% (v/v) with a binding buffer (pH 7.1)
composing 20 mM sodium phosphate, 500 mM NaCl, and 20 mM
imidazole. It was then applied to a HisTrap column (1 mL, GE
Healthcare Bio-Sciences AB, Uppsala, Sweden). The bound proteins
were eluted from the column using a stepwise imidazole gradient
(5 mL fractions using 20, 40, 60,100, and 300 mM imidazole). Active
fractions, which had been eluted using 300 mM imidazole, were
collected. After immobilized metal ion adsorption chromatography
using a HisTrap column, the collected eluent was desalted with a
PD-10 column (GE Healthcare Bio-Sciences AB), pre-equilibrated
with 0.02 M Tris-H2SO4 buffer (pH 8.3).
3.1. Effect of cofactors on peroxidating activity
The enzyme activity of metal-free HPO is usually measured as
the oxidative brominating activity by using an MCD assay. Oxida-
tive bromination reaction proceeds after enzymatic peroxidation
reaction. The resulting value may involve enzymatic and/or non-
enzymatic oxidative bromination. However, non-enzymatic
oxidative bromination's influence can be disregarded under acidic
condition of pH 6.0 or lower because generation of a strongly
brominating active species is not the rate-limiting step under acidic
conditions [14]. Thus, all investigations of carboxylic acids' in-
fluences as cofactors on peroxidating activity of rBPO-A1 were
performed at pH 5.0.
2.2. Activity assay
The following carboxylic acids could be shown to function as
active cofactors during peroxidating activity: acetic acid (AA),
propanoic acid (PA), 1-butanoic acid (BA),1-pentanoic acid (PeA),1-
hexanoic acid (HexA), 1-heptanoic acid (HepA), 1-octanoic acid
(OA), 2-methylpropanoic acid (MPA), 2,2-dimethylpropanoic acid
(DMA), methoxyacetic acid (MAA), 2-chloropropanoic acid (2CPA),
and 3-chloropropanoic acid (3CPA). The following carboxylic acids,
including hydroxyacetic acid (HAA), cyanoacetic acid (CyAA), bro-
moacetic acid (BAA), chloroacetic acid (CAA), dichloroacetic acid
(DCAA), trichloroacetic acid (TCAA), succinic acid (SA), and malic
acid (MA), and amino acids, such as glycine, aspartic acid, glutamic
acid, histidine, lysine, and arginine, were inactive as cofactors. In
conditions of 100 mM AA at pH 5.0 at 25 ꢁC, the specific activity of
rBPO-A1 (18.1 U/mg) was higher than that of BPO-EST from Pseu-
domonas putida (11.7 U/mg) [4]. The specific activities in the pres-
ence of 100 mM PA, MPA, and DMA were 248.6,145.8, and 2.6 U/mg,
respectively. Consequently, PA, MPA, and BA enhanced rBPO-A1's
activity by 13.7-, 8.0-, and 4.6-fold, respectively, compared to that
obtained with AA (Fig. 1A). The relative activity of BPO-EST was
increased 1.18-fold upon substitution of AA to PA as the cofactor,
whereas activity was decreased by 0.60- and 0.40-fold after
The oxidative brominating activity was assayed by measuring
monochlorodimedone (MCD; Fluka Biochemmka, St. Gallen,
Switzerland) disappearance at 278 nm at 25 ꢁC [19] using a UV-
2500PC UV-VIS recording spectrophotometer equipped with a
thermostat (Shimadzu Co., Kyoto, Japan). One unit (U) of oxidative
brominating activity was defined as the amount of enzyme that
catalyzed the consumption of 1
reaction mixture for this assay was composed of 0.06 mg/L rBPO-
A1, 44 M MCD, 10.4 mM H2O2, 300 mM propanoic acid-NaOH
mmol of MCD in 1 min. The standard
m
buffer (pH 5.0), 300 mM NaBr, and 10 mM NaN3. The molar
extinction coefficient (ε) of the enolic MCD anion
(ε ¼ 1.36 ꢂ 104 Mꢀ1 cmꢀ1) [14] was used for the assay because the
stable enol form exists as an enolic anion without the ketoic isomer
at reaction pH [20,21]. The net specific activity of the enzyme in this
assay including a systematic error was obtained by multiplying 1.12
to the obtained value of the apparent specific activity [20].
The molar concentration of the purchased 30% H2O2 solution
(Wako Pure Chemical, Industries, Ltd., Osaka, Japan) was deter-
mined by cerimetric titration using 0.1 M cerium (IV) sulfate solu-
tion (Kanto Chemical Co., Inc., Tokyo, Japan) with 1.5% (w/v) ferroin
Please cite this article as: H. China, H. Ogino, A useful propionate cofactor enhancing activity for organic solvent-tolerant recombinant metal-
10.1016/j.bbrc.2019.06.036
China, Hideyasu
