Flow-through immobilization of Candida rugosa lipase on hierarchical micro-/macroporous carbon monolith

Article abstract of DOI:10.1016/j.molcatb.2011.11.017

Hierarchical micro-/macroporous carbon monoliths are prepared as enzyme carriers for flow-through process. The immobilization of Candida rugosa lipase on micro-/macroporous carbon monoliths is studied. Lipase is immobilized by physical adsorption which lipase solution is circulated through the micro-/macroporous carbon monolith. An accessibility of lipase to the surface inside the micro-/macroporous carbon monolith is enhanced by flow-through method which promotes enzyme-surface interaction and finally leads to rapid enzyme immobilization. After immobilization is conducted for 10 min, the maximum protein binding can be measured. In terms of substrate-immobilized lipase reactions, flowing of substrate through lipase immobilized micro-/macroporous carbon monolith promotes high efficiency in both reaction and product withdrawal. Moreover, at high flow rates of lipase solution in immobilization step, the lipase activity increases. Oxygenated surface of micro-/macroporous carbon monoliths support also demonstrates an interesting effect on lipase immobilization and biocatalyst activity. The initial reaction rate of lipase immobilized on oxygenated surface carbon monolith support has higher activity compared with normal surface.

Full text of DOI:10.1016/j.molcatb.2011.11.017

Journal of Molecular Catalysis B: Enzymatic 75 (2012) 80–85
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Flow-through immobilization of Candida rugosa lipase on hierarchical
micro-/macroporous carbon monolith
Bordin Luangona, Adisak Siyasukhb, Pakorn Winayanuwattikunc, Wiwut Tanthapanichakoond,
Nattaporn Tonanona,∗
a Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10300, Thailand
b Department of Industrial Engineering, Faculty of Engineering, Naresuan University, Pitsanulok 65000, Thailand
c Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10300, Thailand
d SCG Chemical Co., Ltd. I10-1 Rd., Mab TaPhut Industrial Estate, Rayong 21150, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Hierarchical micro-/macroporous carbon monoliths are prepared as enzyme carriers for flow-through
process. The immobilization of Candida rugosa lipase on micro-/macroporous carbon monoliths is studied.
Lipase is immobilized by physical adsorption which lipase solution is circulated through the micro-
/macroporous carbon monolith. An accessibility of lipase to the surface inside the micro-/macroporous
carbon monolith is enhanced by flow-through method which promotes enzyme–surface interaction and
finally leads to rapid enzyme immobilization. After immobilization is conducted for 10 min, the maxi-
mum protein binding can be measured. In terms of substrate-immobilized lipase reactions, flowing of
substrate through lipase immobilized micro-/macroporous carbon monolith promotes high efficiency in
both reaction and product withdrawal. Moreover, at high flow rates of lipase solution in immobilization
step, the lipase activity increases. Oxygenated surface of micro-/macroporous carbon monoliths sup-
port also demonstrates an interesting effect on lipase immobilization and biocatalyst activity. The initial
reaction rate of lipase immobilized on oxygenated surface carbon monolith support has higher activity
compared with normal surface.
Received 13 March 2011
Received in revised form
25 November 2011
Accepted 25 November 2011
Available online 7 December 2011
Keywords:
Lipase
Enzyme immobilization
Flow through
Hierarchical porous carbon monolith
Oxygenated carbon monolith
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
et al. [10] reported that the oxygenated functional group on the
surface of carbon supports prepared by plasma oxidation promotes
Immobilization of enzyme on several supports has been stud-
ied in order to improve the activity, stability, reusability and cost
effectiveness of enzyme [1–3]. The selection of support materials
for enzyme is a very important factor to prevent enzyme leaching
in the process. Porous materials such as carbon, ceramic and poly-
mer are widely used as supports in enzyme immobilization due to
their high surface area and pore volume. Moreover, size and char-
acter of pore structure of these materials can be tailored made to
be specific with enzymes [1,4–7].
For lipase immobilization, the hierarchical porous carbon is an
attractive support. Carbon has hydrophobic surface which most
lipase supports have been modified to be more hydrophobic in
order to improve the activity of lipase [2,8]. Not only hydrophobic
surface but also pore size and shape of the hierarchical porous car-
bon support have a big effect on lipase immobilization and activity
[9]. In addition, functional groups on the surface of carbon sup-
port can be adjusted to suit lipase immobilization. Recently, Quirós
enzyme adsorption and activities of immobilized lipase compared
with normal surface.
Among various shapes of enzyme supports, bead supports
have been commonly used in packed bed or stirred reactor [11].
However, monolithic porous support is an ideal support for flow-
through process. From its low back pressure, good mass transfer
and high throughput [12], monolithic porous support as been used
as a stationary phase in continuous flow process. This material
shows several advantages on many applications such as chromato-
graphic support, separation membrane, effective reaction column
and catalytic carrier [13–17]. Therefore, hierarchical porous carbon
monolith has a great potential as enzyme supports in biocatalyst
reaction under rapid flow conditions to increase the activity and
reduce the reaction time.
In the present study, the immobilization of Candida rugosa lipase
on a hierarchical porous carbon monolith with micropores on an
interconnected macroporous skeleton synthesized from sol–gel
polycondensation of resorcinol and formaldehyde is investigated.
The effects of circulating flows and the flow rates of enzyme and
substrate solutions as well as the effects of oxygenated surface of
micro-/macroporous carbon monoliths on lipase immobilization
∗
Corresponding author. Tel.: +66 2 218 6878; fax: +66 2 218 6877.
E-mail address: nattaporn.t@chula.ac.th (N. Tonanon).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.11.017

B. Luangon et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 80–85
81
are intensively discussed. An interesting point is in most lipase
immobilization on porous materials, only mesoporous or meso-
/macroporous materials are widely used as supports [10,18–20]
while the micro-/macroporous carbon monolith is originally inves-
tigated in this study.
2. Experimental
2.1. Enzyme and chemical reagents
C. rugosa lipase (Type VII) and p-nitrophenyl palmitrate (p-NPP)
are purchased from Sigma–Aldrich Co. Resorcinol (C6H4(OH)2;
99%) is obtained from Fluka. Formaldehyde (HCHO; 36.5%) and
nitric acid (HNO3) are acquired from Ajax Finechem. 1-Butanol
and t-butanol are analytical grades supplied by Carlo erba
Co.
2.2. Synthesis of macroporous carbon monolith precursors
Fig. 1. Experimental set up for flow-through immobilization process by circulating
lipase solution and reaction mixture in immobilization step and enzymatic reaction,
respectively.
Resorcinol–formaldehyde (RF) solutions are prepared from
resorcinol and formaldehyde in de-ionized water. Nitric acid
solution 0.5 M is used as a catalyst in the synthesis of intercon-
nected macroporous carbon monolith [21]. The molar ratios of
resorcinol to formaldehyde (R/F), and resorcinol to water (R/W),
and the mole to volume ratio of catalyst to water (C/W) are
fixed at 0.50 mol/mol, 0.15 mol/mol, and 0.20 mol/ml, respec-
tively. Firstly, resorcinol is dissolved into the de-ionized water
and stirred until the dissolution is completed. Then, the resor-
cinol solution is added to formaldehyde solution and followed
by adding 0.50 M nitric acid. All solutions are then kept in a
thermostated bath. Before RF solutions lost their fluidity, they
are transferred into the cylindrical glass tube where RF gels are
formed. Next, RF gels are aged in hot air oven at 75 ◦C for 24 h.
After aging, RF gels are removed from the tube and dried at 75 ◦C
with hot air until constant weight is achieved. The hierarchical
micro-/macroporous carbon precursor in monolithic form is finally
obtained.
2.5. Lipase immobilization on the hierarchical porous carbon
monolith column by flow-through method
The solution of 1 mg/ml lipases prepared in 20 mM phosphate
buffer solution at pH
7 is circulated through the micro-
/macroporous carbon monolith column by using peristaltic pump
(Fig. 1). The lipase solution is circulated at 10, 22 and 55 ml/min
flow rates for 120 min. After that, the lipase-immobilized carbon
monolith column is three times washed with phosphate buffer to
remove the unbound enzyme. The residual enzyme and washing
solutions are recovered and analyzed for unbound protein. The
lipase-immobilized carbon monolith column is dried in a desiccator
and stored at 4 ◦C.
2.3. Preparation of hierarchical porous carbon monolith column
The protein binding efficiency is calculated as the amount of
protein bound to the carbon monolith column by measuring the
difference of protein between the initial and residual (including the
washing solutions) protein concentrations in solution. The concen-
trations of protein before and after immobilization are determined
by Bradford protein assay method [23].
An activated RF carbon monolith (ACM) is prepared by car-
bonization under 50 cm3-STP flow of N2 gas in a quartz tube reactor.
The sample is heated up to 850 ◦C at 10 ◦C/min heating rate and held
at 850 ◦C for 30 min. While an oxygenated carbon monolith (OCM)
is obtained by CO2 activation which CO2 gas is used instead of N2
gas in carbonization process [22]. Before enzyme immobilization,
the obtained cylindrical shape carbon monolith (8 mm in diame-
ter and 6 cm in length) is clad by heat-shrinkable tubing. Then, the
encapsulated carbon monolith is packed into a precut glass tube
with epoxy glue.
2.6. Enzyme activity assays for lipase-immobilized carbon
monolith
2.4. Characterization of hierarchical porous carbon monolith
Transesterification between p-nitrophenyl palmitate (p-NPP)
and 1-butanol is employed to determine an activity of lipase
immobilized on micro-/macroporous carbon monolith [9]. The
stock solution of p-NPP is prepared in t-butanol. The reaction
mixtures (19 ml p-NPP solution and 1 ml 1-butanol) are circu-
lated through the lipase-immobilized carbon monolith column
at 10 and 55 ml/min flow rates for 180 min at room tempera-
ture. The sampling of 5 ␮l reaction mixture is collected about
20 times within 180 min. Each sampling is then mixed with
300 ␮l triethylamine–ethanol solution (3 ␮l triethylamine in 1 ml
ethanol). The products (p-nitrophynol) formed by the enzy-
matic transesterification are spectrophotometrically determined at
410 nm.
The cross-sectional macropore texture of carbon monoliths
is determined by scanning electron microscope (SEM; JEOL,
JSM-6700F). Macropore size distribution and macropore vol-
ume of the macroporous carbon monoliths are characterized
by mercury porosimeter (Micromeritics, Pore-Sizer-9320). The
micro-/mesoporous properties are obtained from the adsorption
and desorption isotherms of N2 at 77 K measured by adsorption
apparatus (BEL, mini-BEL Sorp.). The surface functional groups
are identified by Fourier transform infrared (FTIR) spectra using
spectrometer recorder (PerkinElmer, 1615). A weight lost after car-
bonization of the obtained carbon monoliths, carbonization yield,
is calculated.

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B. Luangon et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 80–85
Fig. 3. Macropore size distributions of the samples ACM (ꢀ) and OCM (ꢀ).
3.2. Chemical surface property of carbon monoliths
Infrared spectra of carbon monoliths prepared by carboniza-
tion with N2 (ACM) and by physical activation with CO2 (OCM) are
shown in Fig. 5a and b, respectively [24].
Infrared spectra of ACM are analyzed (Fig. 5a). It is clearly seen
that only two peaks can be observed at all spectrum regions. The
peaks at 1624 and 1335 cm−1 can be attributed to C C stretch-
ing vibration and C H bending vibration modes in basal plane,
respectively. Hence, no functional groups on the surface of carbon
monolith prepared by carbonization with N2 (ACM) can be detected
[25–27].
Fig. 2. SEM images show cross-section of the samples ACM (a) and OCM (b).
According to Fig. 5b, four significant peaks which can be
observed at the peaks around 3439, 1630, 1051 and 586 cm−1 in
the sample OCM. The peaks at 1630 and 586 cm−1 can be attributed
to C C stretching vibration modes and out-of-plane bending vibra-
tion of C H, respectively, in the basal plane of carbon. In addition,
the peak around 1630 of OCM is too sharp, it possible presence
of C O vibration. The C OH stretching vibration peak is shown in
3. Results and discussion
3.1. Characterization of porous properties of hierarchical porous
carbon monolith
Macroporous textures of hierarchical carbon monoliths are
observed by SEM images as shown in Fig. 2. These SEM images con-
firm the appearance of interconnected macroporous structure of
the activated carbon monolith (ACM) and the oxygenated carbon
monolith (OCM). Macropore size distributions of carbon monoliths
are shown in Fig. 3. It can be indicated that the macropore diame-
ter of samples ACM is larger than OCM. Moreover, the macropore
volume of OCM is slightly smaller than ACM. More shrinkage in
OCM compared to ACM leads to smaller macropore diameter and
volume in OCM corresponded with carbonization yield. From Fig. 4,
N2 adsorption–desorption isotherms of OCM and ACM are classified
as type I microporous material by IUPAC. Micropores on macrop-
orous skeleton of these carbon monoliths are clearly confirmed by
porous properties from Figs. 3 and 4. According to Table 1, BET spe-
cific surface area (SBET) and micropore volume (Vmic) of OCM are
larger than SBET and Vmic of ACM.
These results indicate that the macropore skeletons of ACM and
OCM are similar as shown by SEM images. However, the macrop-
ore diameter of OCM is smaller compared with ACM whereas the
surface area of OCM is larger. These results indicate that the CO2
activation shows higher shrinkage of the carbon structure than car-
bonization with N2 while higher surface area can be explained by
an increase in micropore volume.
Fig. 4. N2 adsorption–desorption isotherms at 77 K of the macroporous carbon
monoliths prepared by carbonization with N2, ACM and physical activation with
CO2, OCM.

B. Luangon et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 80–85
83
Table 1
Porous properties of the micro-/macroporous carbon monoliths.
Samples
Carbonization yielda
Macropore properties
Micropore properties
Type of isotherm
SBET [m2/g]
Vmacro [cm3/g]
Mean pore diameter [␮m]
Vmicro [cm3/g]
ACM
OCM
57
64
0.65
0.63
18.6
13.5
Type I
Type I
0.19
0.25
289
385
a
Carbonization yield shows mass loss from sample in carbonization process which correspond with the development of microporosity of the carbon [22].
broad band at 1051 cm−1. Moreover this peak is also attributed to
the C C stretching vibrations. Therefore, the presence of peak at
3.4. Effect of lipase solution flow rate and substrate flow rate on
immobilized lipase activity
O
3439 cm−1 can be assigned to the subsistence of hydroxyl or phe-
nol group on the surface of carbon monolith [25–33]. From these
results, there is a high possibility that the oxygenated functional
group on the surface of carbon monolith can be generated by CO2
activation method.
The effects of enzyme solution flow rate and substrate flow rate
are studied by measuring immobilized lipase activity in terms of
initial reaction velocity and protein binding efficiency (Table 2).
The initial reaction velocity which is the rate of product generation
related to the immobilized lipase activity. Although, the immobi-
lized lipase catalyzes transesterification reaction and quantitatively
provides same final cumulative products for all different flow rates,
an increase in the flow rates of lipase solution in the immobilization
process drastically increases the initial reaction velocity.
For the ACM column, the protein binding efficiencies of ACM-10,
ACM-22 and ACM-55 columns are 56%, 58% and 54%, respectively.
It is indicated that flow rate of lipase solution has no effect on the
binding of enzyme on ACM support. However, the initial reaction
velocity of ACM columns is slightly different at various immobilized
flow rates. The maximum initial reaction velocity is obtained from
ACM-55 column immobilized with 55 ml/min flow rate of lipase
solution.
In case of OCM column, OCM-10 immobilized lipase has 71%
protein binding efficiency which is higher than the columns immo-
bilized with 22 and 55 ml/min flow rates (51% and 55% protein
binding efficiencies, respectively), but the initial reaction velocity is
lower. These results suggest that lipase immobilized at 10 ml/min
flow rate has more time for structural rearrangement which is
mainly protein–protein interaction so more protein bindings on
the support lead to steric impediments; therefore, more enzymes
attached on the surface of carbon do not correspond with their
activity [19]. For OCM-22 and OCM-55 columns immobilized with
22 and 55 ml/min flow rates respectively, the initial reaction veloc-
ity on OCM-55 column is 3.3 × 10−4 ␮mol/min which is 2 times
faster than OCM-22 column (1.7 × 10−4 ␮mol/min) while the pro-
tein binding efficiency is the same. It indicates that the high flow
rate of enzyme solution plays a remarkable role in minimizing the
3.3. Flow-through lipase immobilization
The immobilization of lipase is operated by circulating of
enzyme solution through the oxygenated micro-/macroporous car-
bon monolith (OCM) packed in the glass column. The strong
bonding between carbon particles on the skeleton of macroporous
carbon monolith leads to no breakage of small carbon particles after
the circulating flows of lipase and substrate solutions.
Theactivityoflipasesolutionis investigatedwithintimeinterval
to calculate the optimum immobilization time which expressed as
the percentage of residual activity (Fig. 6). A decrease in lipase activ-
ity indicates that the attachment of soluble active enzyme on the
surface of carbon monolith support decreases rapidly with an incre-
ment of immobilization time. The maximum active enzyme loading
is achieved in approximately 10 min of immobilization process at
the lowest of residual activity. High mass transfer and low diffusion
resistance of macroporous carbon monolith strongly help lipase to
rapidly diffuse into the inside surface of hierarchical porous carbon
monolith. Lipase immobilization on micro-/macroporous carbon
monolith by flow-through method results in the shortest immo-
bilization time compared with other diffusion methods.
Fig. 5. FTIR spectra of the carbon monoliths obtained from carbonization with N2,
ACM (a) and physical activation with CO2, OCM (b).
Fig. 6. %Residual activities in enzyme solutions at various immobilization times.

84
B. Luangon et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 80–85
Table 2
Protein binding efficiency and initial reaction velocity of lipase-immobilized on the ACM and OCM columns at various flow conditions.
Columns
Flow conditions in the experiment
Enzyme solution (ml/min)
Protein binding efficiency (%)
Initial reaction velocity (×104 ␮mol/min)
Substrate solution (ml/min)
ACM-55
ACM-22
ACM-10
OCM-55
OCM-22
OCM-10a
OCM-10b
55
22
10
55
22
10
10
55
55
55
55
55
55
10
54
58
56
55
51
71
69
1.0
0.8
0.7
3.3
1.7
1.5
0.6
steric resistance of immobilized lipase in terms of improvement in
enzyme distribution on the surface as a result the substrate eas-
ily reaches the active site of enzyme [10,34]. Therefore, the initial
reaction velocity is increased when the immobilization is carried
out at high flow rate.
As results, the protein binding efficiency of ACM and OCM
columns are similar at 22 and 55 ml/min immobilized flow rates;
although, surface area of OCM is larger. In this case, the attachment
of enzyme on carbon support does not depend on the surface prop-
erties of materials at high immobilized flow rate. However, OCM
column at 10 ml/min flow rate shows more protein loading com-
pared with ACM column. In addition, the chemical and textural on
surface of carbon support has an interesting effect on the initial
reaction velocity. The initial reaction velocity of OCM columns is
clearly higher than ACM columns at all immobilized flow rates.
These results reveal a high possibility that lipase can be induced
to active form when it is attached on oxygenated surface of OCM
column.
The effect of substrate flow rate is studied by flowing of the
substrate through lipase-immobilized carbon monolith column to
catalyze the reaction. The production rate is determined by cal-
culating the initial reaction velocity. The initial reaction velocity
of OCM-10a column measured at 55 ml/min flow rate of sub-
strate is 1.5 × 10−4 ␮mol/min which is 2.7 times higher than
the initial velocity of OCM-10b column (10 ml/min flow rate of
substrate), 0.6 × 10−4 ␮mol/min. As a result, high yield occurs
at higher flow rate of substrate; although, the amount of pro-
tein binding on the supports is similar. It clearly suggests that
the circulating flow of substrate through the lipase-immobilized
micro-/macroporous carbon monolith outstandingly leads to good
substrate–immobilized enzyme interaction when the substrate
flow rate is increased, the products can also be easily removed from
the macroporous skeleton of carbon monolith support. In summary,
the flow rate of substrate in the reaction also plays an important
role on the enzymatic activity because an accessibility of substrate
to enzyme on the surface of micro-/macroporous carbon monolith
support is easy and the production rate is increased at high flow
rate of substrate.
Fig. 7. The relationship between initial velocity and substrate concentration in
transesterification reaction catalyzed by lipase-immobilized on the ACM (᭹) and
OCM (ꢁ) columns.
micro-/macroporous carbon monoliths. The kinetic constant; affin-
ity binding of enzyme toward substrate, K and the maximum
velocity, Vmax can be calculated by using enzymatic equations [35].
For catalytic mechanism, the lipase immobilization on the
OCM and ACM columns show a single-substrate reaction and an
allosteric reaction, respectively [35]. The estimated values of Vmax
and K0.5 of immobilized lipase on ACM column are approximately
2 times lower than values of Vmax and Km obtained from OCM col-
umn (Table 3). Although, the protein binding efficiency of OCM
and ACM columns are similar at high immobilized flow rate, the
carbon monolith with oxygenated surface exhibits clearly higher
efficiency compared with the untreated surface carbon monolith.
Thus, the oxygenated surface of micro-/macroporous carbon mono-
lith exhibits a better transesterification rate. An improvement in
the transesterification rate can probably be explained by an induc-
tion of slightly polar substrate (p-NPP) by the oxygenated surface
of micro-/macroporous carbon monolith.
3.5. Role of oxygenated surface of micro-/macroporous carbon
monolith on kinetic parameters of immobilized lipase
Influence of physical surface treatment of micro-/macroporous
carbon monolith on enzymatic mechanism is discussed. Immo-
bilized lipase on ACM and OCM columns which have no surface
functional group and surface functional groups, respectively are
compared by immobilization at 55 ml/min flow rate. Kinetic
parameters of transesterification activity of immobilized lipase on
micro-/macroporous carbon monoliths are investigated by vary-
ing the initial substrate concentrations of p-NPP (10–50 mM). In
Fig. 7, the plots of initial reaction velocity against the substrate
concentration reveal the catalytic mechanisms of lipase immo-
bilization on the oxygenated surface and untreated surface of
Table 3
Kinetic parameter of lipase-immobilized on the ACM and OCM columns.
Columns
Protein binding efficiency (%)
K (mM)
Vmax (×104 ␮mol/min)
ACM
OCM
50
54
26a
51b
8.3
15.6
a
K is referred to K0.5 since the kinetic results show the sigmoidal curve and a Hill
equation is used to calculate kinetic parameters [35].
b
K is referred to Km since the kinetic results followed the Michealis–Menten
equation [35].

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85
4. Conclusions
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Hierarchical micro-/macroporous carbon monolith is success-
fully used as a support for immobilized C. rugosa lipase by
flow-through method. Flowing of lipase solution through the
micro-/macroporous carbon monolith support is completed within
10 min of the immobilization process resulting from high mass
transfer and low diffusion resistance of macropores, thus enzyme
rapidly access into the surface inside the hierarchical porous car-
bon monolith support. Moreover, the flow rate of enzyme solution
and oxygenated surface are a dominant factor. The activity of
immobilized lipase on ACM and OCM columns is improved with
an increasing flow rate of enzyme solution. In addition, the flow
rate of enzyme solution in ACM column does not have significant
effect on protein binding, but the protein binding on OCM column
is developed at low flow rate. However, the presence of oxygen
on the surface promotes higher efficiency than its activated car-
bon monolith counterpart although the protein binding is slightly
different. Furthermore, the flow rate of substrate in the reaction
plays an interesting role on enzymatic activity since it can lead to
better accessibility of substrate to enzyme on the surface of micro-
/macroporous carbon monolith support. In addition, the products
can be easily removed via macropores of the carbon monolith sup-
port.
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and pore structure can be tailored made. Low back pressure, good
mass transfer and high throughput are advantages of the micro-
/macroporous carbon monolith which can be applied as a support
for flow-through/continuous enzyme reaction or a component in a
micro reactor.
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