Comparison of physical and covalent immobilization of lipase from Candida antarctica on polyamine microspheres of alkylamine matrix

Article abstract of DOI:10.3109/10242422.2014.977266

Polyamine microspheres (PA-M) prepared using polyethyleneimine as matrix were used for the immobilization of Candida antarctica lipase. The isoelectric point of PA-M is 10.6, and the hydrophobicity of PA-M was indicated using naphthalene. Optimization of

Full text of DOI:10.3109/10242422.2014.977266

Biocatalysis and Biotransformation, 2014; 32: 314–326
ORIGINAL ARTICLE
Comparison of physical and covalent immobilization of lipase from
Candida antarctica on polyamine microspheres of alkylamine matrix
Feng Wang1, Ting Ting nie1, Lin Lin Shao1 & Zhenggang Cui1,2
1Department of Chemical Engineering and Technology, School of Chemical and Material Engineering,
Jiangnan University,Wuxi, P. R. China and 2The Key Laboratory of Food Colloids and Biotechnology,
Ministry of Education,Wuxi, P. R. China
Abstract
Polyamine microspheres (Pa-M) prepared using polyethyleneimine as matrix were used for the immobilization of Candida
antarctica lipase.The isoelectric point of Pa-M is 10.6, and the hydrophobicity of Pa-M was indicated using naphthalene.
optimization of conditions showed that the maximal loading of lipase on Pa-M reached 230.2 mg g1 at ph 9.0 and
35°C. an increased buffer concentration had no effect on the activity of lipase but decreased the amount of lipase adsorbed.
Simulation with Langmuir and Freundlich isotherms demonstrated that the adsorption of lipase on Pa-M was thermody-
namically favorable. Covalent crosslinking of the lipase adsorbed extended the ph range and increased the optimal tem-
perature of the lipase activity.The physically adsorbed lipase (P-lipase) and the covalently immobilized derivative (C-lipase)
retained more than 75% and 85% of their initial activity, respectively, after 10 cycles of usage. The half-lives of P-lipase
and C-lipase at 50°C were 15.70 and 27.67 times higher than that of the free enzyme, respectively. Compared to P-lipase,
covalent immobilization obviously reduced the catalytic efficiency and activation energy of the enzyme.
Keywords: Covalent immobilization, electrostatic attraction, hydrophobicity, lipase, polyamine microspheres, physical
adsorption
Introduction
used due to the low cost of inorganic material (Forde
enzyme immobilization is a widely used method to
impart desirable features to enzymes (ansari &
husain 2012; Mateo et al. 2007). Reactions cata-
lyzed by enzymes immobilized on suitable supports
have advantages over those catalyzed by soluble
enzymes such as ease of separation, reusability, and
better stability against inactivation (Bornscheuer
2003). an industrial process should be more eco-
nomical by using immobilized systems (hasan et al.
2006; Mateo et al. 2007; Ranganathan et al. 2008).
Lipases (triacylglycerol acylhydrolases, eC
3.1.1.3) are versatile biocatalysts and are widely used
in the food, pharmaceutical, and chemical industries
(Tan et al. 2010). Various materials have been used
in the immobilization of lipase for improving the per-
formance of the enzyme during industrial operation.
For examples, alkyl modified diatomite, CaCo3,
mesoporous silicate, and activated carbon have been
et al. 2010; ghamgui et al. 2007; Ramani et al. 2010;
Yang et al. 2009); and glyoxyl agarose, chitosan, dex-
trans, cyclodextrin, and alginate were used on
account of their biocompatibility and biodegradabil-
ity (Brigda et al. 2007; Monier et al. 2010; ozmen
et al. 2009; Tahir et al. 2009; Mendes et al. 2013).
Lipases are hydrophilic enzymes with hydropho-
bic regions surrounding the catalytic site (grochulski
et al. 1993). The attachment of lipase to the surface
of a support through hydrophobic interaction has
been widely adopted as a practical immobilization
method (Blanco et al. 2007; gitlesen et al. 1997;
gunnlaugsdottir et al. 1998; Li et al. 2011). it was
also found that hydrophobic interaction between
lipase and supports can promote the activity of
immobilized lipase (Chen et al. 2012; Dong et al.
2013; Jin et al. 2011; Sorensen et al. 2010;Tao et al.
2014). The adsorption of lipases on hydrophobic
all the author are contributed equally to this project.
Correspondence: Feng Wang, School of Chemical and Material engineering, Jiangnan university, Wuxi 214122, P. R. China. Tel: 86-510-82682316. Fax:
86-510-85917763. e-mail: fwang@jiangnan.edu.cn
(Received 24 March 2014; revised 24 June 2014; accepted 13 October 2014)
iSSn 1024-2422 print/iSSn 1029-2446 online © 2014 informa uK, Ltd.
Doi: 10.3109/10242422.2014.977266

Physical and covalent immobilization of lipase from Candida antarctica 315
supports leads to interaction of the hydrophobic
lation of synthesis steps, which greatly limit their
application.
areas surrounding the active site, leaving the active
site exposed to the reaction medium (Kang et al.
2007; Rodrigues et al. 2013). however, a very high
hydrophobicity of the surface has adverse effects on
the adsorption of lipase by limiting access of enzyme
molecules in aqueous solution to the support (Blanco
et al. 2007; galarneau et al. 2006). Polar agents such
as ethanol have been applied to decrease the hydro-
phobicity of supports to improve the adsorption of
lipase (Blanco et al. 2007). electrostatic attraction is
another force driving the adsorption of lipase on sup-
ports (Ye et al. 2007; Yong et al. 2008; Salis et al.
2005). Compared to hydrophobic interaction, the
force of electrostatic interaction is more stable, tight-
ening the attachment of lipase on the support and
reducing leakage during washing and other steps
(Kharrat et al. 2011).To take the advantages of both
two kinds of interaction, Zheng et al. recently devel-
oped a silica particle functionalized with octyl and
sulfonic acid groups to immobilize lipase via hydro-
phobic and strong cation-exchange interaction
(Zheng et al. 2012).The immobilized enzyme exhib-
ited remarkable thermal stability and reusability.
Pei, a polycationic polyamine, is a polymer with
a branched backbone of two carbons followed by one
potentially protonated nitrogen atom. it has the
highest cationic density of any synthetic polymer
currently available (Kitazoe et al. 2005; Kouisni &
Rochefort 2009). Pei has been used to coat the sur-
face of supports to increase the loading of lipase and
improve the stability of the enzyme (Cui et al. 2013;
Feng et al. 2013). however, the physical coatings of
Pei on substrates through electrostatic adsorption
are structurally unstable and detach easily in the
absence of chemical bonds (grunlan et al. 2005; Ji
et al. 2004). Therefore, covalent grafting of Pei on
the substrate has been employed to form stable teth-
ers, avoiding amine leaching (Kassab et al. 2012).
epoxy, aldehyde, and alkyl halide functional groups
have been used to react with functional amine groups
of Pei for the formation of covalent bonds (gao
et al. 2009; Kassab et al. 2012; Wang et al. 2011).
Taking advantage of these covalent reactions, a
method called layer-by-layer (LBL) assembly was
developed for constructing multilayer films of cross-
linked Pei via alternate covalent reactions on sub-
strates such as quartz slides, silicon wafers and
aminosilanized glass surface (hu & Ji 2011; Xia
et al. 2011). in recent years, Pei hollow particles
have been developed from Pei coating microparti-
cles using a core removal method (Sunintaboon
et al. 2006; Tong et al. 2008). however, both t
he LBL and the core removal method are time-
consuming procedures for obtaining a thin shell of
crosslinked Pei and demand sophisticated manipu-
in this study,a polyamine microsphere of alkylam-
ine matrix, Pa-M, was prepared from Pei via Pick-
ering emulsion polymerization. The microspheres
have both a high density of ionizable amino groups
and hydrophobic patches available to interact with
lipase, which ensure both high enzyme loading and
maintenance of the lipase activity. Factors affecting
the activity recovery and properties of the immobi-
lized lipase are discussed.
Materials and methods
Materials
Polyethyleneimine (Pei, Mw70000 Da; 50 wt%
solution in water), lipase from Candida antarctica
type B (1.68 u/mg protein), was purchased from
Sigma-aldrich Shanghai Trading Co. Ltd., Shang-
hai, China. glutaraldehyde (25 wt% solution
in water) and liquid paraffin were obtained from
Sinopharm Chemical Reagent Co., Ltd., Shanghai,
China. Calcium carbonate nanoparticles (40–90 nm
in diameter) were supplied by Shanghai Yaohua
nano-tech Co. Ltd., China. other reagents were of
analytical grade and used as received.The lipase was
purified using a desalting column (econo-Pac 10
Dg, Bio-Rad) before use. Depending on the desired
ph, sodium acetate-acetic acid (ph 4.0–5.0), dipo-
tassium phosphate-potassium dihydrogen phosphate
(ph 6.0–8.0), borate (ph 9.0), or glycine-naoh
(ph 10.0–12.0) buffers were used. Molarity of the
buffer was 25 mmol L1.
Preparation of PA-M via Pickering emulsion
polymerization
80 ml of Pei solution (20 wt%) was mixed with 120
ml of liquid paraffin and 1.44 g CaCo3 nanoparti-
cles and the mixture was homogenized. 150 ml of
25% glutaraldehyde was then added dropwise into
the emulsion at a speed of 0.2 mL min1 using a
syringe pump under moderate magnetic stirring.
after 15 h of reaction at room temperature, micro-
spheres were collected by filtration and rinsed suc-
cessively with diethyl ether, isopropyl alcohol, and
distilled water. Subsequently, under gentle stirring,
the microspheres were reacted with 1 g naBh4 in
100 ml sodium bicarbonate buffer at ph 10 for 2 h
to reduce the imine bonds at room temperature.The
product, CaCo3/Pa-M composite microspheres,
was collected by filtration and rinsed successively
with diethyl ether and water.For obtaining polyamine
microspheres (Pa-M), the CaCo3/Pa-M composite

316 F.Wang et al.
microspheres were soaked in 0.001 mol L1 hCl for
2 h to remove CaCo3 particles. Finally, Pa-M were
collected by filtration and washed with distilled water
until the ph value of the washing water became neu-
tral. Fourier transform infrared spectroscopy analy-
sis of Pa-M was performed. iR (KBr) cm1:
3200–3400 (–n–h stretching), 1550–1650 (–n–h
in-plane deformation vibration), 1046 (–C–n
stretching), 900–650 (–n–h out-of-plane deforma-
tion vibration), 2850–2929 (–C–h stretching), 1465
(–C–h asymmetric bending vibration). illustration
of the preparation procedure of Pa-M is demon-
strated in Scheme 1.
naphthalene to the glass and caps. The head space
in the vials was approximately 30 mL. naphtha-
lene-in-acetone stock solutions were then injected
with a microsyringe so that the initial naphthalene
concentration in the aqueous phase was within the
range of 0.05–1.0 mg L1. The volume fraction of
acetone in the aqueous phase in each vial was kept
less than 0.002 to avoid possible cosolvent effects.
The vials were rotated end-over-end at 20 rpm in
the dark at room temperature. after 3 days, the con-
centration of naphthalene in the head space was
analyzed by gas chromatography (gC) using the
Shimadzu gC-2010 (Kyoto, Japan) coupled with
flame ionization detector. The concentrations of
naphthalene in aqueous phase were then calculated
based on henry’s Law (with henry’s Law constant
for naphthalene at 25°C0.0197 [Xiao & Wiesner
2012]) and the concentrations of naphthalene in
gaseous phase. all the samples were repeated in
triplicate.
Determination of morphology, zeta potential,
hydrophobicity, and thermogravimetry of PA-M
optical microscopy of Pa-M was done using aVhX-
1000 optical microscopy (Keyence Corporation,
Japan) equipped with a digital camera (Fujifilm,
Japan).The morphological structure of microspheres
was examined with an S-4800 scanning electron
microscope (hitachi Ltd., Tokyo, Japan) and the
diameter distribution determined by the method of
Cohen et al.(Cohen et al. 2011). The average diam-
eter was estimated by counting 200 microspheres.
Thermogravimetric (Tg) and derivative thermogra-
vimetric (DTg) analysis were performed on a Tga/
SDTa851e thermogravimetric analyzer (Mettler-
Toledo international inc., Switzerland). Zeta poten-
tials of Pa-M dispersed ultrasonically in the water of
different ph were measured using a ZetaPlus zeta
potential analyzer (Brookhaven instruments Corpo-
ration, newYork, uSa) at 25°C.
The hydrophobicity of the surface of Pa-M was
estimated by measuring its ability to absorb naph-
thalene (Xiao &Wiesner 2012). 0.05 g microspheres
in 10 mL 0.1 M phosphate buffer (ph7.5) sus-
pension were added to a 40 mL sample vial and
capped with Mininert screw-cap valve (Sigma-al-
drich Co. Ltd.). For each sample, a control vial was
set up following the same procedure, while using 10
mL ultrapure water instead of the microsphere sus-
pension, to account for the possible adsorption of
Adsorption of lipase by PA-M
in a 50 mL flask with stopper, 0.05 g of Pa-M was
incubated with 10 mL lipase buffer solution con-
taining 0.1% (v/v)Triton X-100 on a rotary shaker
with an agitation speed of 150 rpm at 25°C. The
adsorbed lipase was separated by filtration and
washed with a buffer solution of the same ph
value as the enzyme solution to remove the
unbound enzyme.The washing solutions were col-
lected for determination of protein content and
enzymatic activity.The amount of enzyme adsorbed
was estimated by determining the amount of
enzyme in the adsorption solution and the wash
buffers. The influence of temperature on the
adsorption was investigated over the range of
25–55°C.The data shown in figures and tables are
the average values and standard errors of experi-
ments repeated three times.The experimental data
on the initial lipase concentration and the amount
of lipase adsorbed were fitted into the Langmuir
and Freundlich adsorption isotherm model using
MicroCal origins.
NH
PEI
NH
CaCO -liquid paraffin
3
N
CH
suspension
N
GA
N
H
C
N
Dissolution of CaCO
3
Crosslink of PEI
particles with HCl
Hydrogenization
Emulsification
with GA
PEI solution
Polyamine microsphere
PEI emulsion droplet stabilized
GA
by CaCO particles
3
Scheme 1. illustration of the preparation of the polyamine microspheres from Pei via Pickering emulsion polymerization.

Physical and covalent immobilization of lipase from Candida antarctica 317
The separation factor (RL) was estimated from
Influence of temperature and pH on the activity
of lipase
the Langmuir adsorption isotherm parameters and
used to estimate the affinity between the lipase and
Pa-M (Weber & Chakravorti 1974).
The optimum operating ph and temperature for free
and immobilized lipases were determined by measur-
ing the activity of the lipase over a temperature range
of 20–60°C and ph from 6.0 to 11.0.The maximum
activity of each set was taken as 100%. The relative
activity (%) was calculated by comparing the activity
of every sample with the maximum activity.
1
RL 
(1)
1KLC0
where C0 is the initial concentration of the lipase
(3.5 mg mL1) and KL is the Langmuir isotherm
constant (mL mg1). The values of RL indicate
whether the isotherm is unfavorable (RL 1), linear
(RL 1), favorable (0RL 1), or irreversible
(RL 0).
Thermal stability and reusability of immobilized lipase
Reusabilities of the immobilized derivatives during the
hydrolysis of olive oil were examined by measuring the
residual activities of immobilized enzymes.The condi-
tions of hydrolysis reaction were based on methods
described previously (abramic et al. 1999; Cho &
Rhee 1993; Liu et al. 2005). 0.05 g immobilized lipase
or 15 mg free enzyme was allowed to react with a
mixture of 4 mL olive oil emulsion and 6 mL phos-
phate buffer (ph 7.5) in 25 mL flasks with stoppers
on a rotary shaker at an agitation speed of 150 rpm at
35°C. after each run the immobilized derivatives were
separated by filtration, washed with the phosphate buf-
fer of ph 7.5, and then reintroduced into another fresh
reaction medium.The olive oil emulsion was prepared
by emulsifying 50 mL olive oil in 150 mL distilled
water containing 4 wt% PVa (Yang et al. 2010).
The thermal stabilities of free and immobilized
lipases were tested at 25°C and 50°C in 25 mmol L1
phosphate buffer of ph 7.5. The reaction mixtures
were transferred into an ice bath every hour and the
residual activities measured. The initial activity of
enzyme was assigned as 100%.The thermal inactiva-
tion constant (kd) and half-life (t1/2) for the lipases
were calculated using eqs. (2) and (3), respectively
(Cabrera-Padilla et al. 2012; olusesan et al. 2011).
The treatment of PA-M adsorbed lipase using
glutaraldehyde
one gram of lipase adsorbing Pa-M was put into a
20 mL solution of 1 wt% glutaraldehyde and incu-
bated for 1 h on a rotary shaker with an agitation
speed of 250 rpm at 25°C. afterwards, the covalently
immobilized lipase was collected by filtration and
washed several times with distilled water and stored
at 4°C.
Protein content and lipase activity determination
The amount of protein in the enzyme solution and
in the washing solution was measured by the Lowry
method (Lowry et al. 1951).
The enzymatic activities of free and immobilized
lipases were measured by detection of the p-nitrop-
henol released by hydrolysis of p-nitrophenyl palmi-
tate (p-nPP) (Chiou & Wu 2004). 0.5 g of p-nPP
dissolved in 100 mL of ethanol was used as sub-
strate.The increase in absorbance at 410 nm caused
by the release of p-nitrophenol in the hydrolysis of
p-nPP was measured spectrophotometrically. Lipase
was added to a mixture of 1 mL of 0.5% (w/v) p-nPP
solution and 1 mL of 25 mmol L1 phosphate buffer
(ph 7.5) at 35°C. after 5 min incubation, the reac-
tion was terminated by adding 2 mL of 0.5 n
na2Co3 followed by centrifuging for 10 min (10,000
rpm).The supernatant of 0.5 mL was diluted 10-fold
with distilled water, and measured at 410 nm using
a Shimadzu 1240 uV-vis spectrophotometer. one
unit (u) of lipase activity was defined as the amount
of enzyme which released 1 mmol p-nitrophenol per
minute under the experimental conditions.The rela-
tive activity (%) was the ratio between the activity of
every sample and the maximum activity of sample.
The specific activity (u⋅mg1 of protein) was calcu-
lated by dividing the enzyme activity by the protein
content.


At
A
(2)
ln
kdt



0 
ln2
t1/2 
(3)
kd
where At is the residual activity after heat treatment
for a period of incubation (u), A0 is the initial
enzyme activity (u), kd is the inactivation constant
(h1), and t1/2 is the half-life (h).
Determination of physicochemical parameters of free
and immobilized lipases
The Michaelis–Menten kinetic parameters of the
free and immobilized lipases were determined from

318 F.Wang et al.
100
80
60
40
20
0
0
20 40 60 80 100 120 140 160 180
Diameter (µm)
Figure 1. Digital photograph and diameter distribution plot of Pa-M.
a Lineweaver–Burke plot.The initial rates of reaction
were measured in 25 mmol L1 phosphate buffer
(ph 7.5) at 35°C. The catalytic efficiency (h) and
the value of turnover number (kcat) were calculated
from the following equation:
with CaCo3 particles, initially producing CaCo3/
Pa-M composite microspheres. after reduction of
iminelinkagesandremovalofthestabilizer,polyamine
microspheres Pa-M were obtained, which appeared
translucent under the microscope (Figure 1). The
diameter of Pa-M ranged from 48.08 to 132.21 mm,
with an average value of 88.25 mm. Scanning elec-
tron microscopy images of Pa-M are shown in
Figure 2.The surface of Pa-M was rough and small
pores were recognizable on the surface. The Tg
analysis of Pa-M is recorded in Figure 3. The total
weight loss of the microsphere was 83.3% from 50°C
to 530°C. according to the DTg curves, Pa-M
shows an initial mass loss over the range from 50°C
to 100°C, due to loss of adsorbed water. Correspond-
ing to an obvious endothermic peak in the range
from 132.5°C to 204°C, the microsphere lost about
6.5% of its weight, possibly owing to evaporation of
small compounds such as residual glutaraldehyde.
The maximal decomposition rate of Pa-M was found
at 440°C and the temperature for total decomposi-
tion of Pa-M was 530°C.
kcat
Vm
η

(4)
Km
E K
[ ]
m
t
where [E]t is the enzyme concentration and Vm is the
maximal velocity.
The activation energies (Ea) of the native lipase
and its immobilized derivatives were calculated
according to the arrhenius law.
Results and discussion
Morphology,TG analysis, surface charge
and hydrophobicity of PA-M
Pickering emulsions are more stable than ordinary
emulsions as they use fine solid powders as the emul-
sifier (Chen et al. 2010; Kralchevsky & nagayama
2000). in this study, Pei was cross-linked with glu-
taraldehyde in liquid paraffin emulsion stabilized
investigation of the surface charge on Pa-M at
different ph showed that the isoelectric point (pi) of
the microspheres was 10.6 (Figure 4a). Thus, the
Figure 2. SeM images of Pa-M.

Physical and covalent immobilization of lipase from Candida antarctica 319
Physical adsorption and covalent immobilization
of lipase on PA-M
0.0
100
80
60
40
20
0
–0.1
The forces driving the adsorption of proteins on sur-
faces include electrostatic attraction, hydrophobic
(B)
–0.2
(A)
interactions, and hydrogen binding.The roles played
by different driving forces during the adsorption
process depend on the specific characteristics of
enzyme and the support surface involved. Changes
in the ph, temperature, initial concentration of
lipase, ionic strength of adsorption medium and the
incubation time on the adsorption of lipase by Pa-M
were investigated and the results are shown in
Table i. When the ph of the solution was 10.6 (pi
of Pa-M), the surface charge of Pa-M would be
reduced to a minimum, and electrostatic interaction
between the lipase and the microsphere decreased to
its lowest level (Salis et al. 2005).The adsorption of
the lipase on Pa-M (65.9%) in this situation would
be mainly driven by hydrophobic interaction. When
6.0ph10.6, lipase and Pa-M had opposite
charges.The adsorption in this case would be driven
by mixed modes of electrostatic and hydrophobic
interaction, resulting in the highest yield of lipase
adsorption (86.2%) at ph 9.0.When the adsorption
was carried out at ph  6.0 and ph  10.6, both
the lipase and Pa-M would have the same charge.
electrostatic repulsive forces should limit the adsorp-
tion of lipase on Pa-M. however, the adsorption of
lipase on Pa-M at ph 5.0 and 12.0 still reached
55.78% and 13.7%. Presumably, adsorption in this
case is driven by hydrophobic interaction overcom-
ing electrostatic repulsive forces between proteins
and materials having the same charge (andersson &
hatti-Kaul 2000). however, the buffering capacity
–0.3
–0.4
–0.5
–0.6
–0.7
100
200
300
400
500
600
700
Temperature (°C)
Figure 3. Tg (a) and DTg (B) thermogram of Pa-M.
surface charge of the microsphere is positive when
ph  10.6 and negative when ph  10.6. Consider-
ing the isoelectric point of the lipase is 6.0, the
enzyme should be adsorbed by Pa-M through elec-
trostatic interactions in the ph range of 6.0–10.6.
The surface hydrophobicity of microspheres was
probed using naphthalene as an indicator (Xiao et al.
2012). according to Figure 4B, the amount of
naphthalene adsorbed by Pa-M increased with the
concentration of naphthalene. Clearly, there were
hydrophobic patches on the surface of Pa-M, which
could interact with the hydrophobic surface of lipase
(Derewenda et al. 1992), promoting adsorption of
the lipase on Pa-M. interaction of Pa-M with the
hydrophobic lid covering the active center of lipase
should facilitate exposure of the active site to the
reaction medium, resulting in the activation of
enzyme (Kang et al. 2007; Rodrigues et al. 2013).
(A)
(B)
30
20
16
12
8
20
10
0
4
6
8
10
12
14
pH
–10
–20
–30
4
0
0.005 0.010 0.015 0.020 0.025 0.030
equilibrium concentration of naphthalene mg L–1
Figure 4. Zeta potential of Pa-M dispersed in water of different ph (a) and the amount of naphthalene adsorbed by Pa-M at various
concentration of naphthalene (B).

320 F.Wang et al.
Table i. effects of ph, temperature, initial concentration of lipase, concentration of buffer solution and incubation time on the adsorption
of lipase on Pa-M.
Lipase
adsorbed (%)
Specific activity
(u mg protein
Relative activity
of lipase (%)
1
)
*
ph
5
6
7.5
9
10.6
12
55.783.18
75.43.57
80.53.80
86.23.92
65.93.41
13.70.61
0.790.03
0.870.03
1.050.02
0.970.04
0.660.02
0.590.01
64.753.79
71.313.44
86.061.90
79.514.12
54.103.03
48.361.69
o
*
Temperature ( C)
25
35
45
55
72.92.81
852.89
86.12.93
87.43.03
0.940.03
1.080.04
1.030.04
0.870.03
77.053.19
88.523.70
84.423.88
71.313.45
Concentration of buffer solution (mmol mL1
)
*
25
50
75
100
150
200
250
84.34.22
83.294.04
77.893.62
72.073.27
68.033.03
65.922.91
63.902.54
1.050.04
1.040.03
1.060.03
1.040.02
1.050.04
1.070.03
1.060.03
86.063.85
85.242.88
86.882.83
85.241.92
86.063.85
87.702.80
86.882.83
*
incubation time (min)
10
20
40
60
56.62.83
75.83.79
80.44.02
82.84.00
83.14.15
84.394.21
78.293.91
83.14.15
79.13.95
1.170.04
1.100.04
1.040.03
1.050.04
1.060.04
1.070.04
0.990.02
1.060.03
0.990.04
96.133.42
90.163.64
85.152.88
86.063.81
87.003.77
87.903.74
81.492.02
87.003.44
81.484.04
80
100
120
140
160
Lipase adsorbed
(mg g1 Pa-M)
Specific activity
(u mg protein1
)
Relative activity
of lipase (%)
initial concentration of lipase (mg mL1
)
0.41
0.82
1.08
1.91
2.6
77.43.25
144.46.06
183.27.69
2108.82
2279.53
2309.66
0.860.02
0.930.03
1.050.04
1.110.05
1.200.05
1.220.05
70.492.32
76.233.22
86.063.81
90.983.82
98.364.12
1004.20
3.56
1
*
The concentration of lipase in solution used was 1.08 mg mL
.
of the Pei matrix can also offset the influence of ph
variation, promoting adsorption at unfavorable ph
values. although the specific activity of lipase
adsorbed at ph 7.5 was 1.08 times higher than that
of lipase adsorbed at ph 9.0, 9.0 was chosen as the
optimal ph for the adsorption of lipase by Pa-M
due to the higher enzyme loading.
investigation of the influence of temperature on
the adsorption of lipase on Pa-M at ph 9.0
(Table i) showed a significant rise in adsorption with
a temperature increase from 25°C to 35°C.Variation
in temperature can have an important influence
on the conformation and activity of proteins (Dill
1990). elevated temperature can expose the inner
hydrophobic core of a folded protein, resulting in
increased hydrophobic interaction (hjerten 1973).
according to the results in Table i, the differences in
lipase adsorption was not statistically significant in
the window of temperature from 35 to 55°C. We
surmise that the adsorption of lipase in this range of
temperature was controlled by an exothermic pro-
cess (Duff & Kumar 2009). Since the maximal value
of specific activity of the adsorbed lipase was obtained
at 35°C, this temperature was chosen as the optimal
temperature for the adsorption of lipase by Pa-M in
the following studies.
The ionic strength of the adsorption medium is
another important factor affecting protein adsorption

Physical and covalent immobilization of lipase from Candida antarctica 321
Table ii. Kinetic constant of Langmuir and Freundlich isotherms
of lipase adsorption on Pa-M.
on supports (Vieira et al. 2011). an increase in salt
concentration can reduce the polarity of the solution
and also partially decrease the surrounding water
structure on the surface of the absorbents and pro-
tein, promoting hydrophobic interaction of protein
with adsorbents (Chen et al. 2003). in contrast, an
increase in ionic strength leads to decreased electro-
static adsorption (Pei et al. 2001), because a compe-
tition between protein and charged molecules in the
buffer solution occurs during the electrostatic adsorp-
tion process. The influence of different salt concen-
trations on lipase loading on Pa-M is shown in
Table i. Lipase adsorption decreased with an increas-
ing salt concentration. although high ionic strength
can weaken the electrostatic interaction between
lipase and Pa-M, the loading of enzyme in 250 mmol
L1 buffer still reached 179.4 mg g1, which was
about 78.8% of the maximum loading. Thus, owing
to the increased hydrophobic interaction in high salt
concentration solution (Chen et al. 2003), Pa-M can
retain a relative high affinity for lipase in an adsorp-
tion medium of high ionic strength. although ions
interfered with the adsorption of lipase, variation in
the concentration of buffer solution showed no effects
on the activity of adsorbed lipase. This result is sim-
ilar to that reported by Lee et al. (2010), where the
activities of immobilized C. rugosa and R. oryzae
lipases were not influenced by the variation of ion
strength when the concentration of buffer solution
was less than 250 mmol L1.
investigation of the incubation time of lipase
adsorption showed that the loading almost reached
equilibrium within 40 min at ph 9.0 and 35°C.The
maximum loading of lipase, about 230.2 mg g1
Pa-M, was obtained at an initial concentration of 3.5
mg mL1 lipase in solution. This loading is approx-
imately equal to that of Candida antarctica lipase
adsorption on functionalized mesoporous silica
(Blanco et al. 2007) and of lipase adsorption on
polypropylene powder (gitlesen et al. 1997) but is
higher than that of Pei-coated supports (Cui et al.
2013; Feng et al. 2013; ondul et al. 2012). Com-
pared to the Pei-coated supports, Pa-M of cross-
linked Pei matrix offers a higher density of amino
groups for interaction with the enzyme.
Langmuir model
Freundlich model
qm
KL
R2
RL
Kf
1/n
R2
0.8592
253.2
21.2
0.9939
0.013
215.3
0.215
Lipase concentration range: 0.41–3.5 mg mL1; incubation time:
40 min; temperature: 35°C; buffer: 25 mmol L1 borate, ph value
of buffer was 9.0.
Where qm is the maximum adsorption capacity of Pa-M for lipase
(mg g1); KL is the Langmuir isotherm constant(mL mg 1); RL
is the separation factor; Kf the Freundlich constant and n is the
Freundlich exponent.
calculated as 0.013, which is greater than 0 but less
than 1, suggesting that a Langmuir isotherm was
favorable. 1/n is a Freundlich constant, indicating
the type of isotherm. When 01/n1, adsorption
is favorable; 1/n1, irreversible; 1/n1, unfavorable
(Vasiliu et al. 2011). as shown in Table ii.The value
of 1/n indicated that the adsorption of lipase by
Pa-M was favorable.
Because of its commercial availability, low cost
and high reactivity, glutaraldehyde has been consid-
ered as one of the most effective protein crosslinking
reagents (Migneault et al. 2004; Richards & Knowles
1968). The aldehyde groups of glutaraldehyde can
react rapidly with amine groups at around neutral
ph (okuda et al. 1991) and is more efficient than
other aldehydes in generating chemically, biologi-
cally, and thermally stable crosslinks (Fernandez-
Lafuente et al. 1995; hwang et al. 2004; nimni et al.
1987). hence, glutaraldehyde remains the reagent of
choice for crosslinking, although many reagents and
newer methods are available (govardhan 1999).
Pa-M carries a high density of amine groups that
can not only be used for anionic binding of lipase
but also can be used as the basis for cross-linking the
enzyme with glutaraldehyde. however, glutaralde-
hyde can cause denaturation of immobilized enzymes
(Romdhane et al. 2011). a low concentration of glu-
taraldehyde is favorable for high activity recovery,
while a high concentration of glutaraldehyde tends
to inactivate the enzyme (Bhandari et al. 2009; Pan
et al. 2011; Park et al. 2002; Wang et al. 2008).
Therefore, the effects of glutaraldehyde treatment on
the activity of lipase adsorbed on Pa-M were inves-
Langmuir model and Freundlich isotherm were
compared in the evaluation of the adsorption process
of lipase on Pa-M as a unit operation. Some model
parameters were determined by nonlinear regression
and shown in Table ii. Comparing the simulation
based on these two models showed that the Lang-
muir equation fitted the experimental data best.
The theoretical maximum adsorption capacity of
Pa-M, 253.2 mg g1, is higher than the experimen-
tal value of maximal loading.according to the results
in Table ii, the value of the separation factor RL was
Table iii. Deactivation constants (Kd) and half-life (t1/2) values
for the free lipase and the immobilized derivatives.
Kd (h1
)
t1/2 (h)
25°C
25°C
50°C
50°C
native enzyme
P-lipase
C-lipase
0.0469
0.0122
0.0105
0.401
14.77
56.8
66
1.73
27.17
51.33
0.0255
0.0135

322 F.Wang et al.
110
100
90
80
70
60
50
40
30
20
10
range for attaining 90% activity of lipase was extended
from 7.2–8.0 (free enzyme) to ph 6.7–8.6 (P-lipase)
and ph 6.4–8.7 (C-lipase).This increased adaptabil-
ity of lipase to variation in ph after immobilization
may be due to the buffering capacity of Pei matrix
of Pa-M (akinc et al. 2005).
Figure 6B demonstrates the temperature-activity
profiles of the native lipase and the immobilized
derivatives under the optimum ph conditions. The
optimum temperature for the activity of lipase
increased from 35°C (the free enzyme) to 40°C
(P-lipase) and 40–45°C (C-lipase). Thus, the opti-
mum temperature for activity was increased by
immobilization. This result is similar to that where
C. antarctica lipase was immobilized on cotton terry
cloth fibrils coated with Pei (ondul et al. 2012).
Running reactions at elevated temperature are
advantageous because of the higher diffusion rate,
lower substrate viscosities, and increased reactant
solubilities (hasan et al. 2006).
0
0.0
0.5
1.0
1.5
2.0
Concentration of glutaraldehyde (%)
Figure 5. effect of the amount of glutaraldehyde addition on the
activity of lipase.
tigated (Figure 5).The activity of immobilized lipase
reached its highest value when 1% glutaraldehyde
was added. any further increase in glutaraldehyde
concentration had a negative effect on the lipase
activity.
Thermal stability and reusability
after adsorption and crosslinking, the specific
activity of the physically adsorbed lipase (P-lipase)
and the covalently immobilized derivative (C-lipase)
was determined as 1.22 u mg protein1 and 1.31 u
mg protein1 separately, which is slightly less than
that of the native enzyme (1.68 u mg protein 1).
immobilization of an enzyme to a support often
limits the freedom of enzyme molecules to undergo
drastic conformational changes, increasing the
stability of enzyme towards denaturation. Figure 7a
shows that the thermal stability of lipase was
increased by immobilization. incubating at 25°C for
8 h, the free lipase retains about 72.5% of its initial
activity, while both P-lipase and C-lipase retain
more than 90% of their initial activities. When the
incubations were conducted at 50°C, the native
lipase lost almost all of its activity, while P-lipase
and C-lipase retained about 79.5% and 90%
of their initial activity, respectively. Thus, Candida
antarctica lipase showed a better thermal stability
Optimum pH and temperature for the operation
of lipase
The activities of the native and immobilized lipase
under different ph conditions are shown in Figure 6.
The ph value (ph 7.5) for optimum activity was not
changed by physical adsorption. however, the ph
(A)
(B)
100
100
90
80
70
60
50
40
30
20
10
0
90
80
70
60
50
40
native lipase
P-lipase
native lipase
P-lipase
30
20
10
0
C-lipase
C-lipase
6
7
8
9
10
11
20 25 30 35 40 45 50 55 60 65
pH
Temperarture (°C)
Figure 6. ph-activity (a) and temperature-activity (B) profiles of the native lipase, P-lipase and C-lipase, respectively.

Physical and covalent immobilization of lipase from Candida antarctica 323
(A)
(B)100
100
90
80
70
80
60
40
20
0
60
25°C native lipase
50
40
30
20
10
0
50°C native lipase
25°C P-lipase
50°C P-lipase
25°C C-lipase
50°C C-lipase
C-lipase
P-lipase
0
1
2
3
4
5
6
7
8
9
0
2
4
6
8
10
Time (h)
Run
Figure 7. Thermal stability of the native and immobilized lipase (a) and reusability of the immobilized derivatives (B).
on Pa-M than that on octyl silica (Blanco et al.
2004). electrostatic attraction and hydrophobic
interactions between Pa-M and lipase comprised
the main driving forces promoting multipoint inter-
action between Pa-M and enzyme, enforcing con-
formational stability of the enzyme. Since covalent
linkages are more stable than physical interactions,
C-lipase showed a higher stability than P-lipase.
The half-life values of the free enzyme, P-lipase and
C-lipase at 25°C and 50°C are shown in Table iii.
at 25°C, the t1/2 of the free enzyme was approxi-
mately 26% and 22.38% of that of P-lipase and
C-lipase respectively. at 50°C, the t1/2 of P-lipase
and C-lipase was 15.70 and 27.67 times higher than
that of free enzyme. Thus, the thermal stability of
the Candida antarctica lipase at the high tempera-
ture was significantly improved upon immobiliza-
tion. The enhanced hydrophobic interactions as
temperature increased could increase the thermal
stability of immobilized lipase at higher temperature
(Cabrera-Padilla et al. 2012). Based on the results
ofTable iii, the values of Kd decreased from 0.0469
interaction. Compared to P-lipase, C-lipase was
more stable and retained more than 85% of its initial
activity after 10 runs. Covalent linkages are more
stable than electrostatic and hydrophobic interac-
tions, limiting configurational changes of enzyme
molecules during operation and reducing leakage of
the enzyme during recycling.
Kinetic constants of free and immobilized lipase
The value of the kinetic constant, Km, can reflect the
affinity of enzymes to substrates. a low value of Km
represents a high affinity between enzymes and sub-
strates. Kinetic constants of the free and immobi-
lized lipase were determined by using olive oil
emulsion as the substrate.The Km and Kmapp for the
free lipase, P-lipase and C-lipase were 5.23, 6.79
and 8.76 mmol L1 respectively.The increased value
of the Michaelis–Menten constants indicates that
the accessibility of substrate to the active sites of the
immobilized enzyme molecules was decreased due
to the steric hindrance caused by contacting with
h
1 (25°C) and 0.401 h1 (50°C) for native enzyme
the support surface (Kang et al. 2005). The values
to 0.0122 h1 (25°C) and 0.0255 h1 (50°C) for
P-lipase, to 0.0105 h1 (25°C) and 0.0135 h1
(50°C) for C-lipase, respectively.The reduced value
of Kd indicates the increased stability of enzyme as
a result of immobilization (olusesan et al. 2011).
The reuse of an enzyme constitutes the main
process advantage of biocatalyst immobilization.
The reuse of an immobilized enzyme permits sim-
plification of the design of the reactor and the con-
trol of the reaction (Kharrat et al. 2011). The
investigation showed that P-lipase retained about
75% of its initial activity at the end of 10 rounds of
recycling (Figure 7B). The physical adsorbed P-li-
pase was quite stable during recycling, due to the
help of the multipoint electrostatic and hydrophobic
of Vm
for P-lipase and C-lipase were 65.76 and
app
66.15 mmol L1 min1, which are respectively 1.19
times and 1.2 times higher than the Vm of the free
enzyme, at 54.95 mmol L1 min1. Since the molec-
ular mass of lipase from Candida antarctica type B
is 33 kDa (uppenberg et al. 1995), the catalytic effi-
ciency (h) of the enzyme was calculated as 0.0289
L mmol1 min1 for the native lipase, 0.0265 L
mmol1 min1 for P-lipase and 0.0234 L mmol1
min1 for C-lipase. Thus, the catalytic efficiency of
lipase reduced about 8.3% (P-lipase) and 19%
(C-lipase) after immobilization. This reduction may
be caused by steric hindrance of the hard surface of
microsphere impeding the access of reacting materi-
als in solution.

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Conclusions
The Zeta potential test and detection of hydropho-
bicity showed that Pa-M microspheres based on an
alkylamine matrix can adsorb lipase through mixed
hydrophobic and electrostatic interactions. after
optimization of conditions, the maximum loading of
lipase was obtained at ph 9.0 and 35°C in 40 min
when the initial concentration of lipase was 3.5 mg
mL1 in 25 mmol L1 buffer solution. evaluation
of the adsorption process by a Langmuir model and
Freundlich isotherm found the adsorption of lipase
by Pa-M was favorable. Compared to P-lipase, the
covalently immobilized lipase (C-lipase) showed bet-
ter ph stability and thermal stability. C-lipase could
retain greater than 85% of its initial activity after 10
rounds of recycling, which was higher than with
P-lipase. The catalytic efficiency of lipase reduced
after both physical adsorption and covalent immobi-
lization, while the activation energy of lipase was
decreased by covalent immobilization, implying
favorable changes in the conformations of the enzyme
molecules during covalent coupling. Pa-M micro-
sphere formed from an alkylamine matrix shows
great potential for academic research and industrial
applications.
Declaration of interest: The authors report no
declarations of interest. The authors alone are
responsible for the content and writing of the
paper.
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