100
I. Plagemann et al. / Journal of Molecular Catalysis B: Enzymatic 87 (2013) 99–104
of this study was to verify and characterize the recombinant
enzyme.
microplate reader (Synergy 2, Biotek, Bad Friedrichshall, Germany),
which was tempered to 22 ◦C (except determination of temperature
optimum).
Enzyme activity was calculated on the basis of the molar extinc-
tion coefficient of the conjugated diene hydroperoxides at 234 nm
(ε = 2.5 × 104 M−1 cm−1). Blanks were carried out using 40 L buffer
instead of enzyme solution. All experiments were performed in
triplicate.
2. Materials and methods
2.1. Chemicals
The purified recombinant enzyme was obtained as described
by Zelena et al. [27]. Tris–HCl (>99%), citric acid monohydrate
(>99%), formic acid (>98%), anhydrous sodium sulfate (>99%) and
disodium phosphate (>99%) were purchased from Roth (Karlsruhe,
Germany); linoleic acid (>99%, hydroperoxide-free) and sodium
borohydride (>99%) were from Sigma–Aldrich (Taufkirchen, Stein-
heim, Germany); boric acid (>98.5%), iron(II) chloride tetrahydrate
(>98%) and LOX-1 from soybean were provided from Fluka (Seelze,
2.5.1. Effect of pH and temperature on enzyme activity
The pH optimum was determined at 22 ◦C using McIlvaine
citrate–phosphate buffer (0.1 M citric acid and 0.2 M Na2HPO4) for
pH 4.0–8.0 and 100 mM borate buffer for pH 8.0–9.5. Determination
of the optimal temperature was performed in the range of 22–50 ◦C
using 182.4 mM citrate-phosphate buffer (pH 7). The addition of
the substrate solution was not carried out until the enzyme-buffer
mixture was tempered at the appropriate temperature for 5 min.
2.2. Protein concentration
2.5.2. Study of kinetic parameters
The protein concentration was determined by the method of
Lowry [28] using DC-Protein-Assay (Bio-Rad, Munich, Germany)
and bovine serum albumin as a standard. In the concentration range
used (0.2 mg mL−1–1.2 mg mL−1) the calibration curve was linear
with a coefficient of determination of R2 = 0.992.
Estimation of the Michaelis–Menten constant (Km) and the max-
imum activity rate (vmax) was based on the method of Lineweaver
and Burk [31] using various substrate concentrations (0.1–1 mM).
Each experiment was carried out with enzyme solutions of three
different concentrations (0.075 ng L−1, 0.1 ng L−1, 0.15 ng L−1).
2.6. Chemical characterization of reaction products
The protein samples were diluted 1:2 with SDS loading buffer
and denatured for 10 min at 95 ◦C. SDS-PAGE was performed
according to [29] using 12% (w/v) polyacrylamide gels. Proteins
were stained with 0.1% (w/v) Coomassie Brillant Blue R-250
(Serva, Heidelberg, Germany). Unstained standard proteins (Bio-
Rad, Munich, Germany) were used for the preparation of a
calibration curve for the determination of molecular masses.
For Western blot analysis the proteins were transferred to a
nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany).
The recombinant dioxygenase was detected using a Penta-His HRP
conjugated Kit (5 PRIME, Hamburg, Germany). Staining was carried
out using 4-chloro-1-naphtol and H2O2 according to the manual
instructions.
To characterize reaction products arising from bioconversion
of linoleic acid, 500 L Tris–HCl buffer (20 mM, pH 7) and 500 L
2 mM linoleic acid solution were incubated with 2 L enzyme solu-
tion (22 g purified enzyme) for 30 min at 22 ◦C on an orbital
shaker. Reaction products were compared to those obtained with
LOX-1 from soybean using the same conditions at pH 10 (borate
buffer, 100 mM).
2.7. Substrate specificity
Activity of the recombinant enzyme toward trilinolein and
methyl linoleate was analyzed by the photometrical assay
described above or LC–MS methods, respectively. The substrate
solution was prepared as described, without addition of NaOH.
2.4. Peptidolytic cleavage of the His-tag
To eliminate the His-tag epitope from the recombinant LOXPsa1,
50 g of the purified protein preparation were mixed with 1 g
Factor Xa Protease (NEB, Frankfurt am Main, Germany) in Factor
Xa-Buffer (Tris/HCl 20 mM, NaCl 100 mM, CaCl2 2 mM, pH 8.0) and
incubated for 6 h at 23 ◦C. A specific inactivation of the peptidase
after the cleavage was omitted.
2.8. Preparation of racemic hydroperoxy linoleic acid
Fenton’s reaction was performed to obtain racemic hydroper-
oxides of linoleic acid as reference compounds. Aqueous solutions
of hydrogen peroxide (9.88 M), FeCl2 (25 mM) and linoleic acid
(25 mM with 8 mM Tween 20) were mixed in a 5 mL volumetric
flask and filled up with distilled water to reach final concentrations
of 100 mM, 0.1 mM and 8.25 mM, respectively. The mixture was
incubated for 20 h at 22 ◦C in the dark.
2.5. Enzyme activity
Enzyme activity was determined spectrophotometrically by
monitoring the increase in the absorbance at 234 nm due to
the transformation of linoleic acid to the respective conjugated
hydroperoxydienes [30]. The assay was carried out in an UV-
transparent 96-well microtiter plate containing 5 L enzyme
solution (0.1 ng purified enzyme L−1 per well) and 175 L citrate-
phosphate buffer (pH 7; except determination of pH optima). The
reaction was initiated by adding 40 L of a freshly prepared 2 mM
substrate solution, which was made of 20 L (18 mg) linoleic acid,
30 L (33 mg) Tween 20, and 60 L 1 M NaOH in a 2 mL volu-
metric flask and filled up with distilled water. This mixture was
diluted with distilled water to the appropriate final concentration
of 2 mM linoleic acid (except kinetic studies). Absorbance at 234 nm
was recorded every 30 s and monitored at least for 20 min using a
2.9. Synthesis of hydroxy linoleic acid
The oxidation products obtained enzymatically or by Fenton’s
reaction were acidified by addition of 1% formic acid and extracted
using 3× 1.5 mL and 3× 5 mL n-hexane, respectively. Combined
extracts were washed with sodium chloride solution and dried
over Na2SO4. Solvent was evaporated under a nitrogen stream and
residue was resolved in 100 L or 2 mL methanol, respectively. To
reduce the hydroperoxides, NaBH4 was added and samples were
incubated for 30 min at 0 ◦C. Subsequently, the mixture was acidi-
fied with 1% formic acid and extracted with 3× 1 mL and 3× 5 mL
n-hexane, respectively. After evaporation of the solvent, enzymatic