2
W. Song et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 1–7
◦
and an attempt was made to shorten the reaction time and solve
the problem of cell permeability without adding any permeability
reagent. The kinetic parameters of the recombinant thermophilic
ARG and the effects of biotransformation conditions were investi-
gated.
Tris–HCl buffer (pH 9.0) in a final volume of 2 mL at 60 C. The
reaction was stopped after 15 min by adding 1.5 M hydrochloride.
The l-ornithine concentration in the mixture was measured as
described by Chinard [15], and l-ornithine monohydrochloride was
used as the standard. One ARG unit corresponds to the amount of
enzyme that could generate 1 mol l-ornithine per minute from l-
arginine by hydrolysis. The protein concentration was determined
by the Bradford method [16], with bovine serum albumin as the
standard.
2
. Materials and methods
2.1. Materials
The expression plasmid pET-28a (+) and the host strain E. coli
2.5. Determination of the kinetic parameters of the recombinant
ARG
BL21 (DE3) were obtained from Novegen (Masdison, WI). The T vec-
tor, restriction enzymes (NcoI and HindIII), T4 DNA ligase, plasmid
mini-preps kit, and agarose gel DNA purification kit were supplied
by TaKaRa Biotechnology (Otsu, Japan). l-Ornithine, l-arginine, and
phthaldialdehyde were purchased from Sigma-Aldrich (St. Louis,
MO).
2.5.1. Determination of optimal pH and pH stability
The optimal pH and pH stability of the recombinant ARG were
determined at a pH range of 6.0–12.0 by using different solutions,
including 50 mM sodium phosphate buffer (pH 7.0, 7.5), 50 mM
Tris–HCl buffer (pH 8.0, 8.5, 9.0), 50 mM glycine–NaOH buffer (pH
9.5, 10.0, 10.5), 50 mM sodium bicarbonate buffer (pH 10.5, 11.0,
2
.2. Expression in E. coli
1
1.5), and 50 mM KCl–NaOH buffer (pH 12.0). For measuring the
The published sequence of the ARG gene from B. caldovelox was
pH stability of the recombinant ARG, the enzyme was incubated
modified by using codon optimization. The synthesized ARG gene
was inserted into the multicopy plasmid pET-28a (+) by introduc-
ing the NcoI and HindIII restriction sites, and then the obtained
plasmid pET28a (+)-ARG was transformed into E. coli BL21 (DE3).
Luria-Bertani (LB) medium (10 g/L peptone, 5 g/L yeast extract,
and 10 g/L NaCl; pH 7.0) was used for seed culture. The seed cul-
at the indicated pH for 12 h at 4 ◦C and then the enzyme activity
was measured as described previously. The relative activities were
expressed as the percentage of maximum enzyme activity.
2
.5.2. Determination of optimal temperature and temperature
stability
The optimal temperature of the recombinant ARG was deter-
◦
ture was incubated on a reciprocal shaker (200 rpm) at 37 C in a
2
0
50-mL flask containing 25 mL of LB medium (supplemented with
.1 g/L kanamycin sulfate) for 6 h. Fermentation was conducted in
a 500-mL flask containing 50 mL of Terrific Broth (TB) medium
mined by measuring the enzyme activity at various temperatures
◦
(30–90 C) as described earlier. To measure the temperature stabil-
ity of the recombinant ARG, the enzyme activity was determined
after incubating it at the indicated temperature for 0.5 h. The activ-
ity of ARG at each time point was normalized as the percent of
enzyme activity at time zero at each corresponding temperature.
(
1
24 g/L yeast extract, 12 g/L peptone, 5 g/L glucose, 2.31 g/L KH PO ,
2 4
◦
6.43 g/L K HPO , and 0.1 g/L kanamycin sulfate; pH 8.0) at 37 C
2 4
and 200 rpm in a rotary shaker. When the optical density at 600 nm
OD600) reached 0.6, IPTG was added to the culture at a final concen-
(
tration of 0.1 mM for induction. The culture of recombinant strain
without IPTG induction was used as the control.
2
.5.3. Effect of metal ions on enzyme activity
To determine the effect of metal ions on the recombinant ARG,
the enzyme activity was measured as described earlier in the pres-
2
.3. Enzyme purification and SDS-PAGE analysis
2+
2+
2+
2+
2+
2+
2+
ence of various metal ions (Cu , Ca , Co , Mn , Ni , Fe , Mg
,
and Zn2+) at a concentration of 0.1 mM. The relative activity deter-
The recombinant ARG was purified by using a facile purifica-
◦
mined in the absence of metal ions at 60 C for 15 min was taken as
tion procedure comprising a heat-treatment step [14]. The intact
cells were collected and resuspended in lysis buffer (100 mM Tris,
1
00%.
5
0 mM MnCl , pH 7.5) and lysed by sonication (power 285 W, ultra-
2
2
.5.4. Determination of Km and Vmax
The kinetic parameters of the recombinant ARG were deter-
phonic 4 s, pause 4 s, total 20 min). After centrifuged at 200,000 × g
◦
for 1 h at 4 C, the obtained supernatant was rapidly heated to and
◦
mined by using reaction mixtures containing variable amounts of
l-arginine (2–40 mM) in Tris–HCl buffer (50 mM, pH 9.0) at 60 C.
The Km and Vmax values were calculated by the Lineweaver–Burk
plotting method.
incubated at 70 C for 15 min. The insoluble material was removed
◦
◦
by centrifugation at 30,000 × g for 15 min at 4 C. Solid ammonium
sulphate was added to the supernatant to 30% saturation and the
◦
solution was centrifuged at 30,000 × g for 10 min at 4 C. The super-
natant was made 50% saturated by further addition of ammonium
sulphate and the solution was centrifuged as before. The pellet
was solubilized in 200 mM Tris buffer (pH 7.5) and centrifuged as
before. The supernatant was dialysed against 20 mM Tris, pH 7.5,
2.6. Production of recombinant ARG in E. coli
The frequently used TB medium was employed to determine the
◦
ARG activity, and the conditions for the expression of the recombi-
nant ARG were examined.
overnight at 4 C. The purified enzyme was obtained after dialysis
and further centrifugation. The SDS-PAGE analysis was performed
with 5% and 10% polyacrylamide gel slabs for concentration and
separation, respectively (Bio-Rad Laboratories, Hercules, CA). After
electrophoresis, the gels were stained with Coomassie brilliant blue
R-250, and the protein bands in the gels were visualized after
destaining the gels in destaining solution.
2.7. Production of l-ornithine by transformation of l-arginine
with ARG
The transformation reaction was optimized under the follow-
ing conditions: 25 mL of 0.1 M carbonate buffer solution (pH 9.0)
in a 250-mL flask on a rotary shaker (150 rpm) at 60 C. To opti-
◦
2.4. Measurement of ARG activity
mize l-ornithine production, different concentrations of l-arginine
2
+
To measure the ARG activity, the reaction mixture contain-
ing 20 mM l-arginine and enzymes was incubated with 50 mM
(120–200 g/L), intact cells (0–21 g/L, wet cell weight), and Mn
− −6
2
(10 –10 M) were employed and the reactions were analyzed.