Immobilization of polyphenol oxidase onto mesoporous activated carbons - isotherm and kinetic studies

Article abstract of DOI:10.1016/j.chemosphere.2007.04.001

Investigations were carried out in batch modes for studying the immobilization behavior of polyphenol oxidase (PPO) on two different mesoporous activated carbon matrices, MAC400 and MAC200. The PPO was immobilized onto MAC400 and MAC200 at various enzyme activities 5 × 10⁴, 10 × 10⁴, 20 × 10⁴, 30 × 10⁴ U l^-1, at pH 5-8, and at temperature ranging from 10 to 40 °C. The intensity of immobilization of PPO increased with increase in temperature and initial activities, while it decreased with increase in pH. Immobilization onto MAC400 followed the Langmuir model while Langmuir and Freundlich models could fit MAC200 data. Non-linear pseudo first order, pseudo second order and intraparticle diffusion models were evaluated to understand the mechanism of immobilization. The free and immobilized enzyme kinetic parameters (K_m and V_max) were determined by Michaelis-Menten enzyme kinetics. The K_m values for free enzyme, PPO immobilized in MAC400 and in MAC200 were 0.49, 0.41 and 0.65 mM, respectively. The immobilization of PPO in carbon matrices was confirmed using FT-IR spectroscopy and scanning electron microscopy.

Full text of DOI:10.1016/j.chemosphere.2007.04.001

Chemosphere 69 (2007) 262–270
www.elsevier.com/locate/chemosphere
Immobilization of polyphenol oxidase onto mesoporous
activated carbons – isotherm and kinetic studies
*
L. John Kennedy, P.K. Selvi, Aruna Padmanabhan, K.N. Hema, G. Sekaran
Department of Environmental Technology, Central Leather Research Institute, Adyar, Chennai 600 020, India
Received 13 September 2005; received in revised form 27 March 2007; accepted 2 April 2007
Available online 1 June 2007
Abstract
Investigations were carried out in batch modes for studying the immobilization behavior of polyphenol oxidase (PPO) on two differ-
ent mesoporous activated carbon matrices, MAC400 and MAC200. The PPO was immobilized onto MAC400 and MAC200 at various
enzyme activities 5 · 104, 10 · 104, 20 · 104, 30 · 104 U lꢀ1, at pH 5–8, and at temperature ranging from 10 to 40 ꢀC. The intensity of
immobilization of PPO increased with increase in temperature and initial activities, while it decreased with increase in pH. Immobiliza-
tion onto MAC400 followed the Langmuir model while Langmuir and Freundlich models could fit MAC200 data. Non-linear pseudo
first order, pseudo second order and intraparticle diffusion models were evaluated to understand the mechanism of immobilization. The
free and immobilized enzyme kinetic parameters (Km and Vmax) were determined by Michaelis–Menten enzyme kinetics. The Km values
for free enzyme, PPO immobilized in MAC400 and in MAC200 were 0.49, 0.41 and 0.65 mM, respectively. The immobilization of PPO in
carbon matrices was confirmed using FT-IR spectroscopy and scanning electron microscopy.
ꢁ 2007 Elsevier Ltd. All rights reserved.
Keywords: Immobilization; Polyphenol oxidase; Mesoporous activated carbon; Adsorption isotherms; Kinetic models
1. Introduction
tion of this biocatalyst in industrial processes, waste
treatment (Wada et al., 1993; Burton et al., 1998), medical
science, biotechnological applications, biological fuel cells
and in the development of bioprocess monitoring devices
like biosensors (Ghindilis et al., 1997; Forzani and Solis,
2000). The production cost of immobilized enzymes is
much lower than that of free enzymes as these can be read-
ily separated from the reaction mixtures and hence can be
used repeatedly and continuously.
Several techniques, mainly based on physical and chem-
ical mechanisms, are applied to immobilize enzymes on
solid supports. Physical methods involve the entrapment
of enzyme molecules within a porous matrix (Duran and
Esposito, 2000; Hussain and Jan, 2000), while chemical
immobilization methods include: (i) enzyme attachment to
the matrix by covalent bonds, (ii) cross-linking between
enzyme and matrix, and (iii) enzyme cross-linking by multi-
functional reagents. Selection of an immobilization strategy
greatly influences the properties of the resulting biocatalyst.
They include several parameters such as overall catalytic
Enzymes are biocatalysts with high specificity, high cat-
alytic efficiency and bio-degradability and hence find major
application in industrial sectors and in medical sciences as
they increase the rate of chemical reaction by lowering the
activation energy (Palmer, 1991). However, their effective
use is restricted because of certain properties like non-reus-
ability, instability, high cost and sensitivity to denatur-
ation. These restrictions, which remain as a challenge for
the application of free enzymes in biotransformations and
chemical processes, are eased by the use of immobi-
lized enzymes. During immobilization, the enzymes are
anchored in a solid matrix so that the enzymes can be
reused and will not be subjected to denaturation. This fea-
ture offers a wider and more economical scope for exploita-
*
Corresponding author. Tel./fax: +91 44 24410232.
E-mail addresses: jklsac14@yahoo.co.in (L.J. Kennedy), ganesanse-
karan@hotmail.com (G. Sekaran).
0045-6535/$ - see front matter ꢁ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2007.04.001

L.J. Kennedy et al. / Chemosphere 69 (2007) 262–270
263
Table 1
activity, effectiveness of catalyst utilization, deactivation
Characteristics of MACs used for immobilization of PPO
and regeneration kinetics and the cost involved. Among
the immobilization procedures, entrapment is the most suit-
able process for ensuring high activity of immobilized
enzymes but sometimes it suffers with the leaching of
enzymes from the matrix (Amsden and Turner, 1999),
owing to the development of weak bonding. The stability
of the immobilized enzymes depends on the strength of
the electrostatic or hydrogen bonds formed between the
support matrix and enzymes. Physical processes are simple
and economically viable.
S. No.
Parameter
Values
MAC400
MAC200
1
2
3
4
5
6
7
8
9
BET surface area (m2 gꢀ1
˚
)
439
35.3
38
218
21.4
48.4
0.70
0.10
50.8
0.69
5.2
Average pore diameter (A)
Carbon (%)
Hydrogen (%)
Nitrogen (%)
2.4
0.50
59.1
0.56
6.6
Ash content (%)
Bulk density (g cmꢀ3
)
Point of zero charge (PZC)
Moisture content (%)
14.3
8.3
Among the available enzymes, polyphenol oxidase (PPO)
finds extensive application in the field of biosensors (For-
zani and Solis, 2000), waste water treatment, removal of
toxic compounds (Wada et al., 1993; Burton et al., 1998),
production of biologically active catecholamines, control
of rancidification process in olive oil (Campenalla et al.,
1999) and production of L-DOPA (Gayatri et al., 2002).
Various solid matrices such as cellulose, collagen mem-
branes, polyacrylamide gel, Enzacryl AA, nylon tubing
(Pialis et al., 1996), glass beads (Estrada et al., 1993),
graphite electrode (Yaropolov et al., 1995), magnetite
(Wada et al., 1992), gelatin gels (Wada et al., 1995), zeolite
(Gayatri et al., 2002), sepiolite (Estrada et al., 1991), ben-
tonite, chitin, chitosan and teflon membrane (Jie et al.,
2004) have been considered for immobilization of PPO.
Most of the studies have concluded that enzymes in inor-
ganic carriers are more stable than those attached with
organic polymers. Previous researchers proved that porous
materials have favoring features for immobilizing PPO
compared to non porous materials owing to their uniform
and adjustable pore size, large surface area, pore volume
and opened structures (Vieira and Fatibello-Filho, 1997).
Therefore, in the present investigation, the feasibility for
immobilization of PPO onto two inorganic mesoporous
carbon matrices, one with high surface area MAC400
and the other with low surface area MAC200, has been
explored. The experimental data were fitted to the non-lin-
ear form of pseudo first order, pseudo second order, and
intraparticle diffusion kinetic models and also to the non-
linear forms of Langmuir, Freundlich and Redlich–Peter-
son isotherm models.
were oven dried at 120 ꢀC for 10 h to drive off the moisture
and stored in glass bottles for further use.
2.2. Reagents
Ammonium sulphate, sodium fluoride, L-Dopa, citric
acid, tri-sodium citrate-2 hydrate and all other chemicals/
reagents were of analytical grade, obtained from E-Merck,
Bombay, India. Commercial grade potato obtained from
the local vegetable market was used as the source for
enzyme extraction.
2.3. Extraction of polyphenol oxidase from potato
The polyphenol oxidase enzyme extract was prepared as
per the procedure reported by Dean (1999) from Solanum
tuberosum (potato). Five hundred grams of washed, peeled
and chopped potatoes was suspended in 500 ml of cold
0.1 M sodium fluoride solution. They were homogenized
using a blender, followed by filtering through a cheese filter.
An equal volume of 50% saturated ammonium sulphate was
added to this filtrate to precipitate PPO. It was allowed to
settle for 2 h and the clear supernatant was siphoned off.
The settled PPO precipitate was centrifuged at 8000 rpm
for 10 min at 4 ꢀC using a Himac C12 22G high-speed
refrigerated centrifuge. The settled solid product was dis-
solved in 0.1 M citrate buffer (pH 4.8) and recentrifuged
at 8000 rpm for 5 min at 4 ꢀC. The supernatant containing
the PPO enzyme was used for further studies.
2. Materials and methods
2.4. Assay of PPO activity
2.1. Adsorbents
PPO activity was determined by measuring the initial
rate of dopachrome formation from the substrate L-Dopa,
as indicated by an increase in absorbance at 475 nm (Duck-
worth and Coleman, 1970) using a Schimadzu UV–Visible
spectrophotometer. One unit of PPO activity was defined
as the amount of enzyme that caused a change in absor-
bance of 0.001 per minute at 25 ꢀC for the sample contain-
ing 2.5 ml of 8 mM L-Dopa solution in 0.1 M citrate buffer
along with 0.5 ml of the enzyme solution. A sample
containing 2.5 ml of citrate buffer with 0.5 ml of enzyme
extract was treated as the blank.
Two mesoporous activated carbons, MAC400 and
MAC200, having different physio-chemical properties as
shown in the Table 1 were selected for immobilization of
PPO. MAC400 and MAC200 were derived from rice husk
using phosphoric acid activation at 800 ꢀC after carboniza-
tion at 400 ꢀC. The preparation of the carbon matrices was
reported by the authors elsewhere (Kennedy et al., 2004).
The mesoporous activated carbon (MAC) of size 600 lm
was washed well with deionised water several times to
remove soluble inorganic compounds. The washed MACs

264
L.J. Kennedy et al. / Chemosphere 69 (2007) 262–270
2.5. Optimization of working parameters
samples were determined using a Leo–Jeol scanning elec-
tron microscope at the desired magnification.
The stable pH range of free enzyme was determined by
evaluating the enzyme activity at different pH values vary-
ing from 3 to 10. The pH of the free enzyme obtained from
single batch extraction was adjusted using HCl/NaOH
solution and the activity was determined at its correspond-
ing pH. The temperature for immobilization was chosen to
be 10, 20, 30, 40 ꢀC and the enzyme activity at which max-
imum immobilization capacity attained was determined
using enzyme activities of 5 · 104, 10 · 104, 20 · 104 and
3. Results and discussion
3.1. Effect of time
The effect of time for immobilization of PPO was carried
out in order to determine the equilibrium points. It was
found that for all experiments, the immobilization was
rapid up to 20 min and then followed an asymptotic
approach to equilibrium around 90 min, as shown in
Fig. 1. Initially the number of adsorption sites available
is higher and the driving force for the mass transfer is
greater. As the immobilization time was increased the num-
ber of bare active sites became less and the PPO molecules
may become clustered inside the carbon particles, thus
impairing the diffusion of PPO. This may be a plausible
explanation for the observed decrease in the immobiliza-
tion capacity after 20 min for both MAC400 and MAC200.
30 · 104 U lꢀ1
.
2.6. Adsorption isotherms
Langmuir, Freundlich and Redlich–Peterson (R–P)
adsorption isotherms were used to determine the immobili-
zation constants. The immobilization isotherms of PPO
onto MAC400 and MAC200 were fitted at four different
pH 5, 6, 7, 8, and at four different temperatures 10, 20,
30, 40 ꢀC. The initial activities of PPO selected were
5 · 104, 10 · 104, 20 · 104 and 30 · 104 U lꢀ1. The immobi-
lization experiment was performed in 100 ml conical flasks
and the contents were agitated at 100 rpm at preset temper-
ature for 90 min in order to attain equilibrium. The mass of
MAC to volume of PPO used was 0.15 g per 10 ml for both
MAC400 and MAC200. The aqueous samples were with-
drawn at preset time intervals and centrifuged, followed
by measuring the PPO activity using a spectrophotometer
at wavelength (kmax) 474, 477, 476 and 476 nm correspond-
ing to pH 5, 6, 7 and 8, respectively.
3.2. Effect of pH
The stable pH for free PPO was screened by determining
its activity in the pH range 3–10. A bell shaped curve was
obtained, showing the PPO activity nearly constant in the
pH range 6–8. The activity of the enzyme was low below
2.7. Determination of kinetic parameters of immobilized
PPO in MAC400 and MAC200
PPO activity measurement was made for different sub-
strate concentrations at constant temperature and pH to
determine kinetic parameters, maximum reaction rate
(Vmax) and Michaelis–Menten constant (Km) for free and
immobilized PPO in MAC400 and MAC200. L-tyrosine
was used as the substrate and the resulting product (L-
DOPA) concentration was determined by the Arnow stan-
dard method (Arnow, 1937). The kinetic parameters were
estimated from the Michaelis–Menten equation,
½SꢁV max
V ¼
ð1Þ
½Sꢁ þ Km
where [S] is the substrate concentration (mM) and V is the
initial reaction rate of the enzyme (mM minꢀ1).
2.8. FT-IR spectroscopy and scanning electron microscopy
(SEM)
The surface functional groups of MAC400 and MAC200
samples were scanned using a Perkin–Elmer FT-IR spectro-
photometer in the spectral range 4000–400 cmꢀ1. The sur-
face morphology of MAC and PPO immobilized MAC
Fig. 1. (a) Activity decay curve for PPO immobilization and (b) PPO
immobilization intensity with respect to time for MAC400 and MAC200
samples. (Conditions: pH 5, temperature 40 ꢀC, activity 5 · 104 U lꢀ1.)

L.J. Kennedy et al. / Chemosphere 69 (2007) 262–270
265
pH 5 and above pH 8. Hence, the effect of pH on immobi-
12
10
8
lization of PPO in MAC400 and MAC200 was studied in
the pH range between 5 and 8. The immobilization effi-
ciency of PPO onto MAC400 and MAC200 with respect
to pH was in the order of 5 > 6 > 7 > 8. The decrease in
immobilization capacity with increase in pH is found to
be influenced by the surface charge on the adsorbent. The
point of zero charge (PZC) value of MAC400 and
MAC200 is 6.6 and 5.2, respectively. At a pH value below
PZC, the carbon adsorbent has a net positive charge and at
pH value above PZC the carbon has a net negative charge.
Therefore, at pH > pHPZC the decrease in adsorption is
observed due to electrostatic repulsion between the nega-
tively charged adsorbent surface and the PPO species.
The immobilization capacity of PPO in MAC400 and
MAC200 at various pH values is shown in Fig. 2.
MAC400
MAC200
6
4
2
0
0
10
20
30
40
50
Temperature (oC)
Fig. 3. Effect of temperature on the PPO immobilization on MAC400 and
MAC200 samples. (Conditions: pH 5, activity 30 · 104 U lꢀ1.)
3.3. Effect of temperature
3.4. Effect of initial enzyme activity
The immobilization capacity of PPO onto MAC400
and MAC200 increased with increase in temperature from
10 to 40 ꢀC as shown in Fig. 3. The increase in immobi-
lization capacity with temperature is an indication of an
endothermic process involved in immobilization of PPO
onto MAC. The increase in temperature may also favor
the altered conformation of already adsorbed PPO on
the active sites of MAC due to the acquired vibrational
energy, making room for further immobilization of new
PPO molecules.
The immobilization capacity of MAC400 and MAC200
increased with increasing PPO activity. At 40 ꢀC, as the ini-
tial PPO activity was increased from 5 · 104 U lꢀ1 to
30 · 104 U lꢀ1, the immobilization capacity increased from
3.48 · 103 to 9.71 · 103 U gꢀ1 for MAC400 and from
0.50 · 103 to 2.23 · 103 U gꢀ1 for MAC200. Although the
immobilization capacity increased with increase in PPO
activity, the percentage of immobilization decreased with
increase in PPO activity. It is observed that at all concen-
trations studied, the immobilization capacity of MAC400
was higher than that of MAC200. This can be attributed
to the difference in pore size and its distribution and the
surface area.
3.5. Equilibrium isotherms
In the present study the experimental data of PPO equi-
librium isotherms were studied at different pH, different
concentration and different temperature, using Langmuir,
Freundlich and Redlich–Peterson models because each
proposed mathematical model assumes a set of hypothesis
to predict the immobilization process.
KLCe
qe ¼
ð2Þ
ð3Þ
ð4Þ
1 þ bCe
qe ¼ KFC1e=n
KRPCe
1 þ bCbe
qe ¼
where qe is the solid phase sorbate concentration at equilib-
rium (U gꢀ1), Ce is the liquid phase sorbent concentration
at equilibrium (U lꢀ1), KL is the Langmuir isotherm con-
stant (l gꢀ1), b is the Langmuir isotherm constant (l Uꢀ1),
KF is the Freundlich constant (U gꢀ1) (l Uꢀ1 1/n
and 1/n
)
is the heterogeneity factor, KRP is isotherm constant
(l gꢀ1), b is the R–P isotherm constant (l Uꢀ1)b and b is
the exponent that lies between 0 and 1.
Fig. 2. Amount of PPO immobilized with respect to pH for MAC400 and
MAC200 samples. (Conditions: temperature 40 ꢀC, activity 5 · 104 U lꢀ1.)

266
L.J. Kennedy et al. / Chemosphere 69 (2007) 262–270
Table 2
Langmuir, Freundlich and Redlich–Peterson isotherm constants for the adsorption of PPO on to MAC200 and MAC400 samples at pH 5
Sample
Temp
(ꢀC)
Langmuir model
Freundlich model
KF ((U gꢀ1
1/n
(l Uꢀ1 1/n
Redlich–Peterson model
Q0 · 102 b · 10ꢀ5 KL
v2
R2
)
v2
R2
KRP
(l gꢀ1
b · 10ꢀ5
(l Uꢀ1 b
b
v2
R2
(U gꢀ1
)
(l Uꢀ1
)
(l gꢀ1
)
)
)
)
)
MAC200
10
20
30
40
18.8
20.3
24.1
27.1
1.44
1.52
2.16
3.33
0.03
0.03
0.05
0.09
0.43 0.998 1.76
0.77 0.994 2.01
0.56 0.991 4.12
0.83 0.994 8.90
0.56 0.50 0.995 0.04
0.52 0.63 0.993 0.06
0.50 0.52 0.996 0.09
0.48 0.72 0.994 0.18
8.69
0.67 0.32 0.998
0.75 0.36 0.997
0.72 0.38 0.996
0.68 0.22 0.992
32.12
96.19
260
MAC400
10
20
30
40
63.2
78.4
89.9
106
7.66
9.92
11.0
21.3
0.48
0.78
0.99
2.25
0.16 0.992 516
0.09 0.998 396
0.07 0.997 417
0.13 0.994 573
0.20 3.61 0.964 0.49
0.11 4.52 0.838 0.77
0.22 6.29 0.812 1.01
0.36 7.45 0.802 2.25
7.78
9.96
11.1
21.3
0.99 0.16 0.992
0.99 0.09 0.998
0.99 0.06 0.997
0.99 0.13 0.994
For all the isotherms studied, the best-fit equilibrium
model was determined based on chi-square, v2 and the cor-
relation coefficient, R2 using non-linear regression meth-
ods. It is observed from Table 2 that the immobilization
data for MAC400 fits the Langmuir model with an average
higher correlation coefficient greater than 0.99, compared
to Freundlich model which has R2 values ranging from
0.80 to 0.96. Also the values of v2 were low compared to
that of the Freundlich model. This suggests that the immo-
bilization of PPO onto MAC400 occurs in a monolayer
form and rules out the possibility of multilayer adsorption.
This is also confirmed by the three-parameter model (R–P)
with regression coefficients above 0.99 and b values equal
to 0.99. Thus, the higher value of b approaching 1 suggests
the Langmuir model of adsorption. The adsorption iso-
therms are shown in Fig. 4. For MAC400 it is observed
that the best fitting models of the experiments are the
Langmuir and Redlich–Peterson isotherms which overlap
each other. The immobilization capacity of MAC400,
was observed to be higher than that of MAC200. The pore
size of the MAC400 having an average pore diameter
˚
35.3 A, enabled the enzyme molecules easy access to the
pores leading to an increased immobilization capacity.
The immobilization of the enzyme molecules onto
MAC400 may involve a series of steps. First, the enzyme
molecules migrate from the bulk phase to the carbon
matrix by diffusion. Second, the enzyme molecule is linked
with unsaturated bonded sites of the mesopore through
electrostatic interaction. The immobilized molecules
undergo compression and refolding so as to accommodate
fresh entries. Finally the enzymes enter the inner sites of
mesopores and get entrapped by establishing bond with
the walls of the pores.
Fig. 4. Langmuir, Freundlich and Redlich Peterson isotherm plots for the
immobilization of PPO on to MAC400 and MAC200 carbon samples.
(Conditions: pH 5, temperature 20 and 40 ꢀC, respectively.)
For MAC200, it is observed that the correlation coeffi-
cient R2 was greater than 0.99 with low chi-square (v2)
for both Langmuir and Freundlich isotherm models. This
suggests the occurrence of both monolayer and multilayer
adsorption of PPO onto MAC. The mixed response for
monolayer and multilayer adsorption is evident from b val-
ues obtained in the range of 0.67–0.75 with regression coef-
ficients above 0.99. The MAC200 has lower adsorption
pore dimension is inadequate for the PPO enzyme molecule
to diffuse into the pores freely. Under circumstances where
the molecular size of enzyme is large enough to enter into
the pores, multilayer adsorption onto outer pore surface
area takes place (Goradia et al., 2005). Multilayer adsorp-
tion is largely confined to the external pore surface area of
the activated carbon and is evident from the SEM photo-
graph of MAC200 and MAC400 immobilized with PPO.
Clustering of the enzyme molecules at the pore entrance
˚
capacity due to the average pore diameter of 21.4 A. This

L.J. Kennedy et al. / Chemosphere 69 (2007) 262–270
267
which could clog the channels so that PPO enzyme is
unable to get into the pores. Thus, it may be concluded that
multilayer adsorption occurs at high concentration and
monolayer adsorption occurs at very low concentration
of PPO.
3.6. Adsorption kinetic modeling
The non-linear forms of the pseudo first order model
(Eq. (5)) (Lagergren and Svenska, 1898), pseudo second
order kinetic model (Eq. (6)) (Ho and McKay, 1998) and
intraparticle diffusion model (Eq. (7)) (Weber and Morris,
1963) were applied to the experimental data at different
temperatures, concentrations and pH.
qt ¼ qeð1 ꢀ expꢀk t
Þ
ð5Þ
ð6Þ
ð7Þ
1
k2q2et
qt ¼
1 þ k2qet
qt ¼ kpt1=2
;
where qe and qt are the amount of PPO immobilized onto
MAC at equilibrium and at time t, (U gꢀ1), k1, k2 and kp
are the pseudo first, second order and pore diffusion rate
constants.
The validity of the order of adsorption processes is
based on two criteria, the first based on the regression coef-
ficients and the second based on predicted qe values. The
first order rate constant k1, the second order rate constant
k2 and pore diffusion constant kp were evaluated with data
fits shown in Fig. 5. The correlation coefficients for the first
order kinetic model for MAC400 and MAC200 were in the
range 0.63–0.96 and 0.80–0.97, respectively. Also the pre-
dicted qe values for both the MAC400 and MAC200
obtained from the first order kinetic model were not very
close to the experimental values as shown in the Table 3.
This indicates that the immobilization of PPO onto
MAC200 and MAC400 does not follow the first order
kinetic model. But for the case of the pseudo second order
kinetic model, it was observed that the correlation coeffi-
cients were greater than 0.99 with the predicted qe values
in good agreement with the experimental qe values, as seen
in Table 3. Thus, it is inferred that the immobilization of
Fig. 5. Pseudo first order, pseudo second order and Weber–Morris plots
for PPO immobilization on to MAC400 and MAC200 carbon samples.
(Conditions: pH 5, temperature 40 ꢀC, activity 5 · 104 U lꢀ1.)
is a plausible explanation for the reduced amount of
immobilization onto MAC200 at higher PPO loading,
Table 3
Comparison of the first and second order rate constants with experimental and predicted qe values for PPO adsorption on to samples (MAC400 and
MAC200) at different pH (conditions: activity: 20 · 104 U lꢀ1, temperature 40 ꢀC)
Parameter
Sample
First order kinetic model
Second order kinetic model
pH
qe · 102 (U gꢀ1
)
qe · 102 (U gꢀ1
(predicted)
)
k1
(minꢀ1
R2
qe · 102 (U gꢀ1
)
k2 · 10ꢀ5
R2
(experimental)
)
(predicted)
(g Uꢀ1 gꢀ1 minꢀ1
)
MAC400
5
6
7
8
130
110
0.06
0.06
0.06
0.05
0.934
0.738
0.632
0.96
129
4.21
3.43
2.72
1.90
0.999
0.998
0.993
0.997
82.0
73.2
61.0
71.3
64.3
58.2
81.1
70.2
60.9
MAC200
5
6
7
8
12.8
11.3
11.0
10.2
11.0
10.1
9.86
7.26
0.05
0.08
0.06
0.05
0.918
0.973
0.967
0.804
12.5
11.0
10.0
9.96
12.1
10.4
9.31
8.49
0.998
0.999
0.997
0.996

268
L.J. Kennedy et al. / Chemosphere 69 (2007) 262–270
PPO onto MAC system belongs to second order kinetic
model.
The intraparticle diffusion model proposed by Weber
and Morris (Weber and Morris, 1963) also did not fully
fit the adsorption data of PPO in both the MACs as it pos-
sessed very low regression coefficients, less than 0.49.
catalytic efficiency is in the order of PPO–MAC < PPO
while the substrate interaction efficiency of PPO–
MAC200 is greater than that of PPO. The efficiencies of
PPO–MAC400 and PPO–MAC200 illustrate that the
enzyme molecule in MAC200 is less strongly held without
affecting the conformation of PPO, while in MAC400 the
enzymes are loaded with altered conformation (strained
condition) leading to retardation of catalytic efficiency.
The catalytic efficiency of the enzyme depends on the rate
at which the active oxy form of PPO is formed from the
meta form. The transformation of the meta to the oxy
through the deoxy form is favored under limited oxygen
supply. This condition is more facilitated in MAC200 as
it is evidenced through the formation of oxy functionalized
groups in MAC200.
3.7. Enzyme kinetic parameters
Kinetic parameters, Km and Vmax for free and immobi-
lized enzymes were calculated from the Michaelis–Menten
plot (Fig. 6). The Km values signify the extent to which
the enzymes have access to the substrates (Shuler and
Kargi, 2005). The lower value of Km represents higher affin-
ity between enzymes and substrates. It is observed that
PPO immobilized in MAC400 (PPO–MAC400) has slightly
lower Km (0.41 mM) value than the free enzyme (0.49 mM)
indicating higher affinity of the PPO–MAC400 towards
substrate tyrosine (enzyme–substrate complex formation)
compared to that of PPO–MAC200, which has Km value
(0.65 mM) higher than others. The higher value of PPO–
MAC200 indicates that enzyme and substrate do not prefer
to come together for a long time. The Vmax values of PPO–
3.8. Shelf-life of enzyme/carbon matrix
To determine the shelf-life of the enzyme/carbon matrix,
the activity of the PPO–MAC400 was checked at an inter-
val of 2 days and continued for 40 days. For PPO immobi-
lized carbons, the enzyme activity remained constant with
small fluctuations, perhaps due to change in conformation
of enzyme (Ahu et al., 2005).
MAC400 and PPO–MAC200 are 0.01 and 0.04 mM minꢀ1
respectively while for the free enzyme it is 0.05 mM minꢀ1
,
.
The low value of Vmax for both the immobilized matrices
indicates the difficulty of enzyme substrate interaction.
The Vmax values are in the order of PPO > PPO–
MAC200 > PPO–MAC400. This suggests that the immobi-
lized PPO in MAC400 has more affinity towards the sub-
strate and the enzyme is less active in catalyzing the
diphenol formation. This is evident from the ratio of
Vmax/Km values for free enzyme, PPO–MAC200 and
PPO–MAC400, with values of 0.11, 0.06 and 0.02 minꢀ1
,
respectively. The greater the Vmax/Km value, the higher
the catalytic efficiency of the enzyme for the substrate.
The kinetic parameters determined suggest that the
Fig. 6. Michaelis–Menten plots of free and immobilized PPO enzyme on
MAC400 and MAC200 samples.
Fig. 7. FT-IR spectrum of (a) PPO, (b) MAC400, (c) MAC200, (d) PPO–
MAC400 and (e) PPO–MAC200 samples.

L.J. Kennedy et al. / Chemosphere 69 (2007) 262–270
269
3.9. Fourier transform infrared spectra (FT-IR)
ance of two bands for PPO–MAC400 and PPO–MAC200
compared to only one band for MAC200 and MAC400.
The appearance of these bands is due to the molecular
interaction of PPO with activated carbon matrices. The
decrease in the broadness of the peak is well observed in
PPO–MAC400 rather than PPO–MAC200 and indicates
that PPO is strongly immobilized in MAC400 as opposed
to MAC200. The band corresponding to 1400 cmꢀ1 due
to C–N stretching vibrations, observed in both the PPO
immobilized carbons, confirms the presence of PPO immo-
bilized in the matrices. These bands were absent in
MAC400 and MAC200 matrices.
The infrared spectra for PPO, MAC400, MAC200,
PPO–MAC400 and PPO–MAC200 are shown in Fig. 7.
MAC400 and MAC200 have a wide band at about 3400–
3425 cmꢀ1 due to O–H stretching mode of hexagonal
groups and adsorbed water. The position and symmetry
of this band at lower wave numbers indicate the presence
of strong hydrogen bonds. For MAC400 and MAC200,
an absorption band is observed at 2920 cmꢀ1 due to ali-
phatic C–H stretching. The broad peak shouldered at
about 1180 cmꢀ1 indicates the presence of phosphorous
content of MAC400. The broad peak shouldered at about
1100 cmꢀ1 and peaks near 800 and 475 cmꢀ1 indicate the
presence of silica for both MAC400 and MAC200 samples.
The FT-IR spectrum of powdered PPO contains two
broad bands near 3200–3400 cmꢀ1 and is due to N–H
stretching frequencies. This can be assigned to primary
amines, as there are two bands owing to N–H asymmetric
and N–H symmetric stretching. A band near 1631 cmꢀ1
corresponds to N–H inplane bending while two sharp
bands at 1400 and 1121 cmꢀ1 correspond to C–N stretch-
ing frequencies. A sharp band at 619 cmꢀ1 corresponds
to the C–H out of plane bending.
3.10. Scanning electron microscopy (SEM)
The surface morphology of MAC400, MAC200, PPO–
MAC400 and PPO–MAC200 matrices are shown in
Fig. 8a–d. The micrographs reveal that MAC400 and
MAC200 are highly porous in nature with differences in
pore size. It is observed that in PPO–MAC200, the PPO
immobilization was minor while in PPO–MAC400, PPO
is well immobilized inside the pores of the carbon matrix.
The enzyme is also located at the outer pore surface area
of activated carbon. This sort of pore penetration of PPO
in MAC400 is not observed in MAC200. This can be
The band near 3135 cmꢀ1 in PPO has been shifted to
3143 cmꢀ1 in PPO–MAC400 and to 3155 cmꢀ1 in PPO–
MAC200, indicating stronger bonding of PPO with the
functional groups of MAC. The N–H stretching frequency
in the region 3200–3500 cmꢀ1 is observed by the appear-
˚
attributed to the decreased average pore size (21.4 A) of
MAC200.
The immobilization of PPO in MAC400 is evident from
Fig. 8c. It is seen in the photo that the enzymes are stuck or
Fig. 8. Micrograph showing (a) surface morphology of highly porous MAC400, (b) MAC200, (c) PPO–MAC400, (d) PPO–MAC200 samples.

270
L.J. Kennedy et al. / Chemosphere 69 (2007) 262–270
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Detailed studies performed with two different carbon
matrices suggested that MAC400 is an ideal matrix for
immobilization of PPO as compared to MAC200. The
optimum conditions for immobilization of PPO onto
MAC400 and MAC200 are temperature 40 ꢀC, pH 5 and
at PPO activity equal to 30 · 104 U lꢀ1. MAC400 followed
the Langmuir mode of adsorption while MAC200 followed
both the Langmuir and Freundlich models, suggesting the
occurrence of both monolayer and multilayer adsorption.
The second order rate equation was followed for the immo-
bilization process. Kinetic parameters for free and immobi-
lized enzymes, determined according to the Michaelis–
Menten equation, showed a lower Km value for PPO–
MAC400 than the free enzyme, indicating higher affinity
towards substrate, while PPO–MAC200 exhibited a higher
Km value. FT-IR spectra provide evidence of the PPO
enzyme immobilized onto the carbon matrices. The PPO
immobilized/carbon morphology was observed in the
SEM photographs.
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Acknowledgement
The first and the other co-authors highly thank the cor-
responding author Dr. G. Sekaran, Deputy Director and
Head, Dept. of Env. Tech, C.L.R.I, for providing financial
assistance and support to carry out the work.
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