1
00
A. Kakoti et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 98–104
were used. Constant 90% ACN with 20% ammonium acetate in
0 min and changes of 15–35% water, 20–0% ammonium acetate
solution and constant 50% ACN in 60–65 min were used followed
and X are the concentration of the respective compounds and area
in the chromatogram, respectively.
6
−
1
by a 5 min equilibration time. A constant flow rate of 0.6 ml min
2.2.7. Determination of kinetic parameters
was maintained during the entire separation process. All mass
were recorded using electro spray ionization in the positive mode
using the conditions: injection volume 10 l; nebulizer gas flow
To determine the Michaelis–Menten constant (Km) and turnover
number (Kcat) of the immobilized enzyme, the activity assay was
done at different concentrations (1–10 mM) of heptanol at opti-
−
N ), 2 l min ; capillary voltage, 4 kV; temperature of the ESI
2
1
(
◦
mum conditions of 30 C and pH 8. Km was calculated using the
◦
◦
probe, 100 C; CDL temperature, 350 C; deflection voltages, 55 V;
detector gain, 1.7 V; collision energy, 5 eV. For scan measurements,
a mass range from 100 to 1000 m/z was chosen. Mass was calcu-
lated based on the calibration of the instrument with standard
leucine enkaphalin using a lock mass m/z 556.2771. The spectra
obtained were processed and analyzed with the MassLynx V4.1
integrated software.
Lineweaver–Burk plot.
3. Results and discussion
The activity of AOx in the cells of A. terreus MTCC 6324 for a wide
range of alcohol substrates was reported by us [14]. We studied here
the molar conversion of various alcohol substrates to their corre-
sponding carbonyl compounds using microsome-bound AOx from
A. terreus as shown in Table 1. The formation of the product aldehy-
des was measured based on the molar absorption coefficient of the
hydrazone derivatives of the carbonyl compounds and the respec-
tive products were confirmed by the mass spectrometry. One of the
representative mass spectra of hydrazone derivative of the product
n-heptanal is shown in Fig. 1. The yields of the carbonyl compounds
were found to be relatively high when long chain primary and sec-
ondary alcohols substrates (C7 and above) and cyclohexanol were
used as substrates. Unlike most of the AOxs reported so far, the AOx
isolated from A. terreus showed poor enzymatic activity towards
short chain alcohol (methanol and ethanol) substrates (Table 1).
Notably, the enzyme is predominantly induced during the growth
of the fungus on long chain alkane substrates [14]. The AOx is cat-
alytically stable against in situ generation of H O , since catalase
2.2.5. Enzyme immobilization on polyurethane foam
Commercially available polyurethane foam matrix was cut into
small equal cubes weighing ∼2 mg each. The cubes were thoroughly
washed in warm deionized water and dried completely by incu-
◦
bating at 50 C for overnight. The foam matrix was immersed in
−1
the enzyme solution (8 mg ml ) for 1 h and then air dried. The
unbound protein was removed by washing the foam matrix in 3 ml
of THB (pH 8.0) under gentle stirring conditions for 30 min. The
washing step was repeated three times for 30 min each and the
◦
foam matrix was stored at 4 C until further use. The amount of AOx
immobilized was calculated from the difference in protein concen-
tration used initially to dip the matrix and the amount that was
leached out during the washing steps. The loading efficiency of the
foam was determined in terms of unit AOx immobilized per mg of
matrix. A control experiment was done using BSA instead of AOx
protein preparation and the protein loading efficiency was deter-
mined. All protein estimations were performed following standard
Bradford method using BSA as standard [17].
2
2
or other H O2 scavenger was not required to be added to the reac-
2
tion mixture to maintain the catalytic activity of the enzyme. It
is reported that the inhibitory effect of H O2 formed during AOx
2
catalysis is generally neutralized by supplementing catalase to the
reaction mixture, without which the oxidase is rapidly denatured
by hydrogen peroxide [18].
2
.2.6. Immobilized AOx catalysis and product analysis
About 16.2 U (∼3.6 mg) of immobilized AOx protein was added
We utilized the catalytic property of the AOx for the production
of n-heptanal, an industrially important compound, from corre-
sponding n-heptanol using the microsomal enzyme preparation
immobilized in to a commercially available polyurethane foam
matrix following a simple adsorption technique. The morphological
characteristics of the AOx immobilized foam matrix were observed
under the optical microscope (Nikon eclipse Ti) (Fig. 2). The pieces
of foam matrix were cut into thin sections to avoid overlapping
images of different layers present in the foam. The immobiliza-
tion may be inferred from the opacity developed on the surface
of the matrix (Fig. 2B) when compared to the control matrix with-
out AOx protein (Fig. 2A). The immobilizing efficiency of the foam
matrix (∼2 mg) was compared between test sample (AOx protein)
to the final reaction mixture vial containing various concentration
of n-heptanol (10–40 mM) in THB (pH 8.0) with the final volume
being maintained at 10 ml and incubated at 30 C and 80 rpm. All
◦
the reactions were done in triplicates and standard error was cal-
culated. After 24 h of the reaction time, the AOx immobilized foam
matrices were separated from the reaction mixture and washed
with THB, pH 8.0 (3 × 10 ml) to remove the loosely bound sub-
◦
strates and products and finally stored at 4 C for the next cycle
of reaction. The reaction mixture containing the carbonyl prod-
uct was transferred to a separating funnel and extracted thrice
(
2 ml each) with chloroform. The organic phase was collected, dried
−
1
by passing through activated Na SO4 and then concentrated by
vacuum rotary evaporator (Equitron) at 80 C. The concentrated
product was membrane filtered and analyzed by GC (Varian 450)
equipped with a CP-Sil 5 CB capillary column. The run parame-
ters for the GC experiment are as follows: carrier gas, N ; flow
rate of N , 1 ml min ; injection port and detector port tempera-
tures, 220 C and 250 C, respectively; oven temperature: 60–250 C
programmed at 10 C min rise. The calibration curves for the sub-
strate n-heptanol and the product n-heptanal were plotted in the
and control protein (BSA) using similar concentration (8 mg ml
)
2
◦
of the proteins in the solution. The AOx immobilized foam matrix
had a protein loading efficiency of 76.6%, while the control BSA
had a protein loading efficiency of 6.7%. The high AOx protein
loading efficiency has been attributed largely to the lipophilic
nature of the enzyme composite consisting of the microsomal
membrane assembly. The microsome-bound AOx preparation used
in this investigation promotes its strong adhesion by means of
large lipoidal aggregation to the porous hydrophobic architec-
ture of matrix. Moreover, the air patches present in the matrix
may act as safe hydrophobic regions which probably prevent the
enzyme from leaching out to the nearby hydrophilic phase. BSA
protein being highly soluble in aqueous phase (maximum solubil-
2
−
◦
1
2
◦
◦
◦
−1
−1
concentration range of 10–100 mM ml . The concentrations of n-
heptanal and the residual n-heptanol were obtained by measuring
the peak area obtained for standard n-heptanol and n-heptanal
chromatograms. To measure the residual substrate and product in
the reaction medium the calibration curves for both n-heptanol and
n-heptanal were constructed acquiring the equations y = 14,836 · X
−
1
ity of 40 mg ml
and fractionated at 75% ammonium sulphate)
was readily leached out from the foam matrix during the wash-
2
2
−1
(
R = 0.995) and y = 29,517 · X (R = 0.991), respectively, where, Y
ing steps. The enzyme loading was calculated to be ∼2.02 U mg