F.A. Muñoz-Guerrero et al. / Catalysis Communications 77 (2016) 52–54
53
2. Materials and methods
2.1. Chemicals
Hydrogen peroxide (30% w/w) and 2-methyl-2-propanol (2M2P)
were obtained from Merck KGaA (Darmstadt, Germany). Styrene
and m-substituted derivatives were purchased from Sigma-Aldrich
(St. Louis, MO). Chloroperoxidase from C. fumago was obtained and
purified as previously reported [22]. The enzyme preparation contained
6530 U mL−1, with a RZ = 1.0 and an enzyme concentration of
289 μmol L−1. All other chemicals, unless otherwise stated, were sup-
plied by Sigma-Aldrich.
2.2. Enzyme activity and determination of catalytic parameters of CPO
Peroxidase activity of CPO was determined spectrophotometrically
using guaiacol as substrate at pH 6.0 and monitoring the absorbance
increase at 470 nm. The transformation rate was estimated by using
an extinction coefficient of 26,600 M−1 cm−1, as guaiacol is converted
to tetraguaiacol [23].
Epoxidation reactions were performed in a 60 mM phosphate buffer,
pH 6.0, containing substrate (0.10 to 10.0 mM), 15% (v/v) co-solvent
(2M2P) and 800 nM CPO final concentration. The reaction was started
by addinghydrogen peroxide solution (40 mM) at a rate of 0.2 μmol/min
flow until reach 1 mM of final concentration (saturating concentration).
The reactions were stopped after 5 min by adding 1 mL acetonitrile
and rapid cooling in ice/water bath. Then, the reaction mixtures were
analyzed in a Knauer high-resolution liquid chromatography (HPLC)
equipped with Smartline 2850 Photodiode Array Detector (HPLC-PDA).
The elution was performed with a mobile phase of 65:35 (v/v)
acetonitrile-water, with a flow of 1.0 mL min−1, through a reverse
phase C18 column 5 μm Eurospher 100–5 (250 × 4.6 mm). The detection
wavelengths were set at 207 nm and 216 nm to measure the generated
diols and epoxides, respectively. All extracts obtained from different ex-
periments were filtered using 0.22 μm nylon syringe filters (Membrane
Solutions), prior to their analysis by HPLC. Enzyme assays and other
UV–VIS experiments were performed with an UV–VIS spectrophotom-
eter UV2310II (Techcomp).
Fig. 1. Specific activity of CPO for the epoxidation of styrene (♦), m-aminostyrene (▲) and
m-chlorostyrene (●) derivatives. The pointed lines correspond to the fit of experimental
data to Hill's equation.
3. Results and discussion
The enzymatic activity of CPO in the epoxidation of m-subtituted
styrenes was evaluated at pH 6.0. It is well known, that CPO is more ac-
tive as halogenase at low pH, while the peroxidase activity is favored at
pH 6.0 [27]. In most of cases, the kinetic data fitted a sigmoidal behavior
as can be observed in the Fig. 1. Styrene derivatives with electron-
withdrawing substituent showed lower catalytic activities than those
derivatives with electron-donor substituents (Fig.1), as in the cases of
m-nitro and m-amino styrene derivatives, respectively. The experimen-
tal data fit the Hill equation from which the catalytic constants were
determined (Table 1). The substrate cooperativity to the enzyme (n)
and the “substrate affinity” (K′) are also shown in Table 1. In the cases
where n was equal to 1.0, the Hill equation becomes the rectangular hy-
perbolic equation and therefore K′ became the Michaelis–Menten con-
stant (Km) corresponding to the substrate concentration that yields
half-maximal velocity.
Prior to catalytic tests, calibration curves for each substrate and their
corresponding epoxides were obtained. Hill's equation (Eq. (1)) was
used as adjustment model to determine the catalytic parameters of
CPO-catalyzed epoxidation of styrene derivatives [24]
ꢀ
ꢁ
n
ν
Et
½Sꢀ
¼ kcat
ð1Þ
n
K0 þ ½Sꢀ
where v is the reaction rate calculated as total concentration produced
of epoxides per minute, Et is the total concentration of enzyme and kcat
represents the catalytic rate constant. The initial concentration of sub-
strate is defined by [S], n corresponds to the substrate cooperativity to
the enzyme and K′ is a constant including different interaction factors
and the intrinsic dissociation constant KS of enzyme-substrate com-
plex. CPO specific activity is defined as v/Et ratio, expressed in min−1
units.
The nitro derivative showed the lower catalytic constant (13 min−1),
whereas the amino derivative showed the highest kcat value
(3869 min−1). A deeper quantitative analysis was performed. Initial-
ly, the kinetic data was correlated with σ-Hamett constant, since
previous report has showed a linear correlation between σ constant
Table 1
2.3. Computational methods
Catalytic parameters of CPO epoxidation of m-substituted styrenes obtained from Hill's
equation and substrate ionization energies (IE) calculated at UB3LYP/6–311(2d,p) level
of theory.
DFT calculations were performed at UB3LYP/6–311 + g(2d,p) level
of theory [25] for all styrene substrates. Geometry optimizations were
carried out without symmetry restrictions for both neutral and ionic
species. Ionization energies (IE) were evaluated as the energy difference
between the radical-cation and neutral species, according the following
equation: S → S+ + e− since has been demonstrated that B3LYP func-
tional give precise estimations for both electronic and energetic proper-
ties of organic compounds [26].
m-Substituent kcat
(min−1
K′
n
Hill model
IE
)
(Moln L−n
)
adjustment (R2) (eV)
H
NO2
Cl
CH3
NH2
90
13
25
58
6
0.7 0.2
6.7 1.2
0.39 0.04 1.2 0.2 0.981
0.26 0.03 2.0 0.2 0.985
1.1 0.1 0.999
1.2 0.2 0.994
8.159
8.762
8.302
7.974
7.109
2
1
1
3869 309 0.42 0.08 0.9 0.1 0.992