Reversible immobilization of cephalosporin C acylase on epoxy supports coated with polyethyleneimine

Article abstract of DOI:10.3109/10242422.2016.1168814

In this study, a recombinant cephalosporin C acylase (CCA) was covalently or physically immobilized on an epoxy-activated support LX1000-EPC4 (EP) or its derivatives, EP-polyethyleneimine (EP-PEI) and EP-ethylenediamine (EP-EDA) with cationic groups on the surface. Zeta potential was used as a tool for activated carrier analysis and immobilization analysis. The EP-PEI (the cationic polymer PEI grafted support) showed higher zeta potential than EP-EDA (the small diamine EDA modified support) and EP support. Among these three supports, immobilization of CCA on EP-PEI had the highest specific activity according to the range of enzyme loadings. Michaelis constant Km values of EP-PEI-CCA and EP-EDA-CCA were 22 mM and 30 mM, respectively, which were lower than that of the free enzyme (43 mM), suggesting that the supports zeta potential is related to the affinity of the enzyme for the substrate. The enzyme immobilized on EP-PEI showed a much higher thermal stability (stabilization factor of 32-fold compared with the free enzyme) than that on the EP-EDA (stabilization factor of 5.5-fold) and EP supports (stabilization factor of 1.7-fold). The adsorption of CCA on EP-PEI support was very strong and reversible. The CCA could be thoroughly desorbed using a high concentration of NaCl (e.g., 2 M) at low pH value (pH 3.0). The regenerated EP-PEI support could then be reused for enzyme immobilization.

Full text of DOI:10.3109/10242422.2016.1168814

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Reversible immobilization of cephalosporin
C acylase on epoxy supports coated with
polyethyleneimine
Yanmei Wei, Hui Luo, Yanhong Chang, Huimin Yu & Zhongyao Shen
To cite this article: Yanmei Wei, Hui Luo, Yanhong Chang, Huimin Yu & Zhongyao Shen
(2016): Reversible immobilization of cephalosporin C acylase on epoxy supports coated with
polyethyleneimine, Biocatalysis and Biotransformation, DOI: 10.3109/10242422.2016.1168814
To link to this article: http://dx.doi.org/10.3109/10242422.2016.1168814
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Date: 10 April 2016, At: 17:51

Biocatalysis and Biotransformation, 2016; Early Online: 1–10
RESEARCH ARTICLE
Reversible immobilization of cephalosporin C acylase on epoxy
supports coated with polyethyleneimine
YANMEI WEI1,2, HUI LUO1, YANHONG CHANG2, HUIMIN YU3 & ZHONGYAO SHEN3
1School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, PR China,
2School of Civil and Environmental Engineering, University of Science and Technology Beijing, Beijing, PR China,
3
and Department of Chemical Engineering, Tsinghua University, Beijing, PR China
Abstract
In this study, a recombinant cephalosporin C acylase (CCA) was covalently or physically immobilized on an epoxy-activated
support LX1000-EPC4 (EP) or its derivatives, EP-polyethyleneimine (EP-PEI) and EP-ethylenediamine (EP-EDA) with
cationic groups on the surface. Zeta potential was used as a tool for activated carrier analysis and immobilization analysis.
The EP-PEI (the cationic polymer PEI grafted support) showed higher zeta potential than EP-EDA (the small diamine EDA
modified support) and EP support. Among these three supports, immobilization of CCA on EP-PEI had the highest specific
activity according to the range of enzyme loadings. Michaelis constant Km values of EP-PEI-CCA and EP-EDA-CCA were
22 mM and 30 mM, respectively, which were lower than that of the free enzyme (43 mM), suggesting that the support’s zeta
potential is related to the affinity of the enzyme for the substrate. The enzyme immobilized on EP-PEI showed a much higher
thermal stability (stabilization factor of 32-fold compared with the free enzyme) than that on the EP-EDA (stabilization
factor of 5.5-fold) and EP supports (stabilization factor of 1.7-fold). The adsorption of CCA on EP-PEI support was very
strong and reversible. The CCA could be thoroughly desorbed using a high concentration of NaCl (e.g., 2 M) at low pH
value (pH 3.0). The regenerated EP-PEI support could then be reused for enzyme immobilization.
Keyword: Cephalosporin C acylase, polyethyleneimine, reversible immobilization, zeta potential
Introduction
When properly designed, immobilized enzymes
often have greater thermal and operational stability
than the soluble form of the enzyme (Garcia-Galan
et al. 2011; Barbosa et al. 2013). In most industrial
biocatalytic processes, the advantages of immobilized
enzymes over soluble enzymes arise from their
enhanced stability and ease of separation from the
reaction media, leading to significant savings in
enzyme consumption (Tran and Balkus 2011;
Barbosa et al. 2013; Rodrigues et al. 2013).
Cephalosporin C acylase (CCA) is a novel enzyme
that directly hydrolyzes cephalosporin
7-aminocephalosporanic acid (7-ACA),
C
to
key
a
intermediate in the production of semi-synthetic
cephalosporin antibiotics (Pollegioni et al. 2013).
Although 7-ACA is currently produced by a two-step
enzymatic process with D-amino acid oxidase and
glutaryl acylase, there are some inherent problems,
for example, the deleterious effect of hydrogen
peroxide, the byproduct of D-amino acid oxidase
catalysis, on enzyme stability, and product yield
Among the traditional enzyme immobilization
protocols, physical adsorption (Daunert et al. 2007;
´
Filho et al. 2008; Lopez-Gallego et al. 2012) and
´
(Lopez-Gallego et al. 2008; Volpato et al. 2010).
covalent bonding (Mateo et al. 2000; Garcia-Galan
et al. 2011) are often used. Enzyme immobilization
by means of physical adsorption (usually via ionic or
hydrophobic interactions) is a mild process with high
retention of activity (Mateo et al. 2006; Marquez
CCA shows great potential for industrial 7-ACA
production because of the advantages of simplicity,
high efficiency, and low cost (Volpato et al. 2010;
Pollegioni et al. 2013).
Correspondence: Hui Luo, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, PR China.
Tel: +86 1082377396. E-mail: luohui@ustb.edu.cn
(Received 17 February 2014; revised 8 May 2015; accepted 17 March 2016)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2016 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.3109/10242422.2016.1168814

2
Y. Wei et al.
et al. 2008; Cantone et al. 2013; Jesionowski et al.
2014). However, enzyme desorption from the sup-
ports (e.g., ionic exchangers) is a crucial issue to be
considered at high ionic strength and pH extremes
interactions between enzyme and support, could be
a diagnostic tool for binding efficiency in enzyme
immobilization studies (Schultz et al. 2008).
Surprisingly, there are few reports considering the
zeta potential of the supports let alone the zeta
potential of the immobilized enzyme in the process of
immobilization (Ding et al. 2005; Gomez et al. 2012).
CCA is a useful enzyme for the industrial
production of 7-ACA. The stability and cost of the
immobilized enzyme are therefore vital for its appli-
cation. In previous reports, CCA was covalently
immobilized on epoxy-activated supports and the
CPC catalyzing efficiency of the catalyst was verified
(Boniello et al. 2010; Zhu et al. 2011). Nevertheless,
with CCA being a heterodimeric enzyme, which is
probably deactivated during catalysis, further efforts
are needed to improve stability and economic aspects
of biocatalysts to meet industrial requirements
(Cantone et al. 2013; Luo et al. 2014; He et al.
2015). Based on the analysis given above, the revers-
ible immobilization of enzymes on PEI coating
supports might increase biocatalyst stability and
simultaneously decrease the cost of supports due to
re-use of the support matrix (Torres et al. 2002).
In this work, ion exchange supports were derived
from the commercial epoxy-activated support LX-
1000EPC4 and used for CCA reversible immobil-
ization. To study the immobilization of CCA, zeta
potential was used to assess protein binding on
different supports and characteristics of the immobi-
lized enzymes. Reversible immobilization of CCA on
the supports and their reusability was also investigated.
´
(Filho et al. 2008; Lopez-Gallego et al. 2012).
As an alternative to physical adsorption, immo-
bilization via covalent bonding irreversibly attaches
the enzyme with no protein leakage during enzyme
utilization; biocatalysts often have good stability and
reusability due to the strong covalent bonds between
the enzyme and support (Mateo et al. 2007a;
Barbosa et al. 2014). For example, immobilizing
enzymes via a two-step mechanism using epoxy-
activated supports, can increase conformational sta-
bility by favoring additional multipoint covalent
attachment of the enzyme (Mateo et al. 2000;
Mateo et al. 2007a). Nevertheless, enzymes immo-
bilized via covalent bonding may undergo conform-
ational changes, which affect the active site due to the
severe reaction conditions during immobilization.
This often leads to a relatively low activity recovery
rate (Mateo et al. 2000; Zhu et al. 2011).
Several protocols for reversible immobilization of
proteins have been described (Batista-Viera et al.
1991; Fuentes et al. 2004; Daunert et al. 2007;
´
Lopez-Gallego et al. 2012). The adsorption of
enzymes on ion exchangers is the most common
and easy protocol for reversible immobilization with
facile regeneration of the supports (Pessela et al.
2006). However, immobilization of enzymes on
traditional ion exchangers is often weak and most
proteins are released from the carriers if the pH value
changes or the ionic strength increases (Filho et al.
2008; Liu et al. 2012).
Unlike traditional ion exchangers, solid matrix
grafted with cationic polymers (e.g., polyethylenei-
mine, PEI) has been proposed as a possible support
for strong but reversible immobilization of enzymes
(Pessela et al. 2006). PEI is a branched polymer with
many amino groups, and supports grafted with PEI
offer a three dimensional surface for interaction with
enzymes via multipoint adsorption (Pessela et al.
2004; Torres et al. 2006). Thus, grafted PEI with a
high charge density enables carriers to adsorb
enzymes more strongly than the usual ion exchangers
and prevents enzyme desorption (Pessela et al. 2003).
The adsorption of an enzyme to a support
depends not only on the nature of the chemical
interactions formed, but also on the surface charge of
the support. Zeta potential measures the charge on
the surface of solid powder particles in suspension,
and this value of surface charge is useful for
understanding and predicting interactions between
particles in suspension (Yukselen and Kaya 2003;
Yukselen-Aksoy and Kaya 2011). Therefore, the
zeta potential, which quantifies the electrostatic
Materials and methods
Materials
CPC and 7-ACA were kindly supplied by North
China Pharmaceutical Co. Ltd. (Shijiazhuang, PR
China). The epoxy support LX-1000EPC4 (EP)
(with an epoxy group content around 60 mmol/g, the
particle size of 150–300 mm, the average pore size
around 15 nm) was kindly supplied by Sunresin
New Materials Co. Ltd. (Xi’an, PR China).
Polyethyleneimine (PEI, Mw 20,000) was purchased
from Aladdin Industrial Inc. Other reagents were of
analytical grade purchased from the local market.
Preparation of CCA
CCA was prepared from cultivation of recombinant
E. coli BL21(DE3)/pET28-CPCAcy (Zhu et al.
2011). The harvested cells were suspended in
sodium phosphate buffer (0.02 M, pH 8.0) and cell
extracts were prepared by sonication and centrifuged.

Reversible immobilization of cephalosporin C acylase
3
Then the 6 histidine-tagged CCA was purified to get
purity higher than 90% by means of immobilized
metal ion affinity chromatography (IMAC) as
described previously (Zhu et al. 2011). The specific
activity of the loaded CCA solution was 9.2 U/mg.
with addition of a suitable amount of CCA solution,
and the mixture kept under mild stirring for 30 min
(Rocchietti et al. 2004; Mateo et al. 2007b). Then
the preparation was washed with sodium phosphate
buffer (0.1 M, pH 8.5) and stored at 4 ꢀC for
further use.
Assay of CCA activity
Detection of zeta potential
The enzyme activity of CCA against CPC was
measured according to a previously described method
(Zhu et al. 2011). One unit of CCA activity is defined
as the amount of enzyme capable of producing 1 mmol
of 7-ACA per minute at 37 ꢀC and pH 8.5.
To meet the particle size limitation of the device for
zeta potential measurement, different supports were
prepared by grinding them to the fine particles and
passing them through a 325-mesh sieve, and then the
same immobilization procedure was performed
according to the method described in section 2.5 and
2.6. The zeta potential of the different CCA prepar-
ations and supports was measured with a ZetaPlus
instrument (Brookhaven Instruments Corporation,
Holtsville, NY).
The specific activity of the immobilized enzyme
was determined under the previously described con-
ditions and was defined as 1 mmol of 7-ACA produced
per minute per gram of wet immobilisate. All data are
the average value of at least three experiments. The
experimental error was less than 5%.
Preparation of supports by modifying EP with PEI
and ethylenediamine (EDA)
Thermal stability of different CCA preparations
Enzyme preparations were incubated at 45 ꢀC in 0.02
M at pH 8.0 phosphate buffer. Samples were
periodically withdrawn, and their activities tested as
described. The CCA residual activity was expressed
as a percentage of the initial activity at the given
incubation time.
Modification of the epoxy support EP with PEI was
performed as previously reported, and a derivative
named EP-PEI was obtained (Rocchietti et al. 2004).
The epoxy support was also modified as previously
described with a small diamine EDA to obtain the
derivative EP-EDA, which is very similar to the
traditional cationic exchanger (Mateo et al. 2007b).
Desorption of enzyme from different ionic supports
Immobilization of CCA on EP support
The immobilized CCA (the CCA preparations with
enzyme loading of 100 U/g support) was incubated in
5 mM sodium phosphate buffer (pH 8.0) with
increasing concentration of NaCl at 25 ꢀC, and
aliquots of the suspension were periodically collected
for activity analysis of the desorbed enzyme.
The epoxy support LX-1000EPC4 was suspended in
1.25 M sodium phosphate buffer at pH 8.0 and
25 ꢀC. A suitable amount of CCA solution was added
to give a final concentration of sodium phosphate
around 0.85 M and the immobilization mixture was
kept under mild stirring for 24 h. Then the derivative
was filtered and washed with sodium phosphate buffer
(0.1 M, pH 8.5) and stored at 4 ꢀC for further use.
Finally, the remaining epoxy groups were blocked
with 3 M glycine in pH 8.0 buffer at 25ꢀC for 24 h
(Mateo et al. 2002). To produce multipoint attach-
ment of CCA on the epoxy support, the EP-CCA
preparation was treated under conditions (in an pH
10 phosphate buffer containing 0.1 M 7-ACA and
20% glycerol, at 25 ꢀC for 24 h), similar to that
applied in the reference for the immobilization of
penicillin G acylase (Mateo et al. 2000).
Kinetic parameters of the CCA preparations
The initial reaction rates of CPC hydrolysis with
free and immobilized CCA (the CCA preparations
with enzyme loading of 100 U/g support) were
determined at different CPC concentrations
(4–20 mg/ml). The Michaelis constant (Km) and
maximum reaction velocity (Vmax) for the free and
immobilized enzyme were calculated according to
Lineweaver-Burk plots.
Reusability of EP-PEI support
Immobilization of CCA on EP-PEI and EP-EDA
supports
To investigate the reusability of the EP-PEI support,
the CCA was desorbed from the EP-PEI support
with 2 M NaCl (pH 3.0) for 30 min at 25 ꢀC
(Wu et al. 2012), and then the regenerated support
The EP-PEI or EP-EDA supports were suspended in
5 mM sodium phosphate buffer at pH 8.0 and 25 ꢀC

4
Y. Wei et al.
Table 1. Zeta potential of different CCA and different supports.
Zeta
was reused for a new immobilization procedure
according to section ‘‘Immobilization of CCA on
EP-PEI and EP-EDA supports’’. In the repeated
adsorption test, the CCA-loading activity in each
cycle was 100 units per gram support.
Sample
Medium
potential(mV)
Free enzymea
CPC-Nab
EP
Buffer (0.1 M, pH 8.5)
Buffer (0.1 M, pH 8.5)
Deionized water
Buffer (0.1 M, pH 8.5)
Buffer (0.1 M, pH 8.5)
Deionized water
Buffer (0.1 M, pH 8.5)
Buffer (0.1 M, pH 8.5)
Deionized water
Buffer (0.1 M, pH 8.5)
Buffer (0.1 M, pH 8.5)
À10.98
À10.06
À0.57
0
EP
EP-CCAc,f
EP-EDA
EP-EDA
EP-EDA-CCAd,f
EP-PEI
À11.59
0.25
0.39
Results and discussion
À9.21
0.57
0.65
Determination of zeta potential
EP-PEI
EP-PEI-CCAe,f
À8.56
To investigate the impact of zeta potential on the
interaction of the support and the enzyme during
immobilization, CCA was covalently or physically
immobilized on different supports, such as EP, EP-
PEI, and EP-EDA. EP-EDA and EP-PEI were
derived from EP by modifying it with the small
molecule EDA and cationic polymer PEI, respect-
ively. It should be pointed out that the particles were
ground and sieved to less than 50 mm to meet
the requirement of particle size limitation of the
device for Zeta potential measurement. The ground
particle should have the same properties as the initial
support for enzyme immobilization, except particle
size. Thus, to obtain the zeta potential data repre-
sentative of the unbroken support, enzyme immobil-
ization on the ground support was performed under
the same conditions of protein loading and immo-
bilization time. Thanks to the increased surface area
of the particle and reduced mass transfer limitation,
the ground support gave a higher immobilization
yield and specific activity than the unbroken one
(data not shown). Though the particle size would
affect immobilization parameters, the different zeta
potentials of ground supports prepared under the
same conditions can be used for comparison of
immobilization differences for different supports.
The zeta potential of different supports and CCA
preparations with small particle sizes is shown in
Table 1. The zeta potential of CCA was found to be
À11.28 mV in pH 8.5 phosphate buffer, indicating
that CCA has a net negative charge when used in
catalysis or immobilization. The substrate CPC has a
similar negative charge in pH 8.5 solution.
aCCA: Concentration of 1 mg/ml.
bCPC: Concentration of 25 mg/ml.
cEP-CCA: CCA was covalently immobilized on EP.
dEP-PEI-CCA: CCA was ionically adsorbed onto PEI-EP.
eEP-EDA-CCA: CCA was ionically adsorbed onto EP-EDA.
fThe CCA loading amount in the immobilization was 100 unit
enzyme per gram support.
a negative charge via electrostatic interaction in
solution.
After immobilization of the negatively charged
CCA, all three supports carried negative charges.
Although the binding of proteins on the carrier
surface is supposed to cover the particle, the groups
on the particle surface still impact the zeta potential
of the final preparation. Besides the material and
support features, the enzyme loading, and even
contributions of part of the protein surface not
involved in the immobilization (which depends on
enzyme orientation) might affect the zeta potential
´ ´
(Ryan and O’Fagain 2007; Serra et al. 2013). Due to
the polycationic PEI coating on the support surface,
the EP-PEI preparation also had the highest zeta
potential after immobilization, followed by EP-EDA.
The difference in zeta potential of these supports
might affect the enzymatic properties of their CCA
preparations in some way (Meder et al. 2013;
Honary and Zahir 2013).
Immobilization of CCA on different supports
As shown in Figure 1A, the immobilization yield of
CCA on the EP, EP-EDA, and EP-PEI supports was
nearly 100% when the enzyme loading was low, but
decreased gradually with increasing enzyme loading.
The immobilization yield of EP-PEI was lower than
EP and a little higher than EP-EDA with higher
enzyme loading, while the enzyme activity and
apparent activity recovery of EP-PEI were higher
than that of EP and EP-EDA. The higher zeta
potential of EP-PEI did not result in a higher
immobilization yield. This might be because the PEI
coating on the support reduces the pore diameter or
even blocks the pores, and thus the final available
surface area for enzyme absorption on EP-PEI is
The hydrophobic polyacrylate-based epoxy sup-
port was electrically neutral in phosphate buffer.
The EP-PEI and EP-EDA, derivatives of the EP
support, however, carried a positive charge because
of the cationic groups from the modifying reagents.
Since zeta potential is a function of the surface
charge of a particle and the nature and composition
of the surrounding medium in which the particle
is suspended, the high density of amino groups on
the surface of EP-PEI resulted in the highest zeta
potential of the three supports. Therefore, the EP-
PEI support should easily adsorb an enzyme carrying

Reversible immobilization of cephalosporin C acylase
5
(A)
(B)
100
80
60
40
20
60
40
20
0
0
30
60
90
120
150
0
30
60
90
120
150
Enzyme loading amount (U/g)
Enzyme loading amount (U/g)
(C)
80
60
40
20
0
30
60
90
120
150
Enzyme loading amount (U/g)
Figure 1. Immobilization of CCA on different supports. (A) Effect of enzyme loading on the immobilization yield. (B) Effect of enzyme
loading on the immobilized activity. (C) Effect of enzyme loading on the recovery rate of activity. Symbols: (g) EP; ( ) EP-EDA; and (m)
ꢁ
EP-PEI. The immobilization yield is defined as the ratio of the adsorbed enzyme to the initial amount in the solution. The apparent activity
recovery is the ratio of the assayed activity of immobilized enzyme to the theoretical activity of adsorbed enzyme.
smaller (Chen et al. 2013; Lin et al. 2013). The EP
support had a higher immobilization yield than the
ionic supports EP-EDA and EP-PEI. However, the
covalent bonds between enzyme and EP support
appeared to distort the enzyme structure, leading to a
lower activity yield (Mendes et al. 2011; Zhu et al.
2011).
ionic strength buffer to achieve the first step of
immobilization, physical adsorption of the enzyme
on the support (Barbosa et al. 2013). And the second
step of immobilization process, covalent binding of
the enzyme to the epoxy support, normally lasts up to
24 h (Zhu et al. 2011; Barbosa et al. 2013).
EP-PEI adsorbed more protein than EP-EDA
with the same enzyme loading and had a higher zeta
potential (Figure 1A, Table 1). This suggests that the
polycationic PEI had a more pronounced impact
than the small molecule EDA on zeta potential of the
immobilized enzymes. Though the PEI grafted
supports usually display a relatively low loading
capacity for enzymes (Liu et al. 2012), the EP-PEI
preparation exhibited the highest apparent activity,
and activity recovery (Figure 1A–C). Thus, this
cationic polymer coating support seems to be a
suitable carrier for CCA immobilization with less
activity loss, especially when the purified enzyme
with high specific activity was loaded.
Thermal stability of different CCA preparations
CCA is a protein with heterogeneous subunits with
relatively poor stability during use. As shown in
Figure 2, free CCA was not stable when incubated in
solution at 45 ꢀC, and its stability correlated with
protein concentration suggesting that this multimeric
protein tends to dissociate in solution (Bolivar et al.
2006; Rocha-Martin et al. 2009). Thus, stabilization
of the CCA quaternary structure is a goal of immo-
bilization, because the dissociation of multimeric
enzymes often strongly correlates with enzyme inacti-
vation (Bolivar et al. 2009; Garcia-Galan et al. 2013).
As expected, all the immobilized enzymes showed
better stability than their soluble counterparts
(Figure 3). Figure 3 shows that the CCA immobi-
lized on EP-PEI had the best stability and its activity
declined very slowly at 45 ꢀC. The enzyme immobi-
lized on the cationic support EP-EDA had a half-life
of 100 min at 45 ꢀC and pH 8.0, a value much lower
Furthermore, the positive zeta potential of EP-PEI
and EP-EDA enables them to immobilize the
enzyme via electrostatic interaction, which can be
achieved in a very short time and at low salt
concentration. In contrast, enzyme immobilization
on the hydrophobic epoxy support EP requires a high

6
Y. Wei et al.
support is able to establish a non-distorting and
very strong ionic interaction with the protein (Torres
et al. 2005).
100
80
60
40
20
0
It was shown that the CCA covalently immobi-
lized on the epoxy support had a low thermostabi-
lization factor of 1.5-fold compared with the free
enzyme, a value even lower than that of EP-EDA
immobilisate (Figure 3). Immobilisates on epoxy
support have been reported to have improved stabil-
ity when immobilization is performed at a higher pH
value or by blocking the remaining epoxy groups
with highly hydrophilic compounds (Mateo et al.
2002; Barbosa et al. 2013). To reduce the hydro-
phobicity of the support surface, the remaining epoxy
groups of the support were blocked with 3 M glycine.
This derivative had unchanged activity but a 17%
increased thermal stability. The EP-CCA prepar-
ation was also treated at alkaline pH value (pH 10) to
produce further covalent binding between enzyme
and support. However, the activity of derivative
decreased by 23% although the thermal stability
slightly increased by 14%. It seems that these
protocols for stabilization of the enzyme immobilized
on epoxy support, which are effective for a lot of
enzymes (Mateo et al. 2000; Mateo et al. 2002), were
less effective for CCA.
0
10
20
30
40
50
60
Time (min)
Figure 2. Inactivation courses of free CCA at different concen-
trations. Symbols: (g) 0.01 U/mL; ( ) 0.1 U/mL; (m) 0.65 U/
ꢁ
mL; and (n) 1 U/mL. Experiments were carried out at 45 ꢀC in
100 mM sodium phosphate buffer pH 8.0.
100
80
60
40
20
0
Since the e-amino group of lysine residues on the
protein surface plays an important role in covalent
binding of the enzyme on epoxy or aldehyde
activated supports, the number of lysines is crucial
for a multi-point covalent binding (Serra et al. 2013).
There are nine lysine residues in CCA but only two
of them are located on the larger b-subunit, resulting
in weak stabilization of the whole enzyme structure
(Luo et al. 2014). The very limited and uneven
distribution of surface lysine residues might explain
why the epoxy support cannot produce a very stable
immobilized CCA. Previously published results also
suggest that increasing enzyme stability via a few
strong covalent bonds is not as good as tridimen-
sional multi-point ionic adsorption (Torres et al.
2005). In our recent work, the covalently immobi-
lized enzyme could be further stabilized by cross-
linking with amines (e.g., PEI), which is somewhat
similar to stabilization of the tridimensional structure
by the cationic polymer PEI in this work (He et al.
2015). However, the covalent immobilization was
irreversible so the support could not be reused.
0
1
2
3
4
Time (h)
Figure 3. Thermal stability of different CCA preparations. The
treatment was performed at pH 8.0 and 45 ꢀC. Symbols: (g)
Soluble enzyme; (m) Enzyme immobilized on EP support; (
)
ꢁ
Enzyme immobilized on EP-EDA support; and (n) Enzyme
immobilized on EP-PEI support. The residual activity is expressed
as a relative percentage of the original activity.
than on EP-PEI. To quantify the thermal stabiliza-
tion of immobilized enzyme, the stabilization factor
(the ratio of half-life times between immobilisate and
soluble enzyme) was calculated. Stabilization factors
of enzyme immobilized on EP-PEI and EP-EDA
were 35.6-fold and 5.5-fold, respectively.
Although multipoint interactions would occur in
the binding of CCA to both EP-EDA and EP-PEI via
ionic exchange, the interactions between the enzyme
and the EP-EDA support would be relative weak on
the ‘‘plane’’ surface (Pessela et al. 2003; Filho et al.
2008). However, the EP-PEI support was provided a
flexible cationic polymer coated surface, allowing a
greater percentage of the protein surface to interact
with the support via a ‘‘tridimensional adsorption’’
(Torres et al. 2002, 2006). Thus, the EP-PEI
Desorption of enzymes from different ionic supports
Uncontrolled desorption of an enzyme from its
support during use is a disadvantage of immobiliza-
tion via physical adsorption (Filho et al. 2008;
´
Lopez-Gallego et al. 2012). To investigate this, the

Reversible immobilization of cephalosporin C acylase
7
enzyme immobilized on EP-PEI and EP-EDA sup-
ports was incubated at different salt concentrations
to assess the interaction strength between the enzyme
and the support. Enzymes on both the EP-PEI and
the EP-EDA desorbed gradually from the support
when the ionic strength increased. However, the
residual activity of EP-PEI immobilized CCA was
higher than CCA immobilized on EP-EDA in all the
solutions with different ionic strength (Figure 4).
Around 70% of CCA remained on EP-PEI when
treated with 1 M NaCl solution. The results
indicated that the enzyme was more strongly
adsorbed on EP-PEI, and the flexible grafted PEI
polymer is thought to play an important role. The
PEI grafted supports provide a three dimensional
surface for interaction with enzymes via multipoint
ionic adsorption, where the enzymes may penetrate
in the polymeric bed formed by flexible long chains
of PEI (Torres et al. 2006). Thus, the adsorbed
enzymes are less likely to release from the support
during application.
Generally, Michaelis constant Km is used to
evaluate the enzyme-substrate affinity, and a lower
Km value means a higher affinity of substrate to the
enzyme. The CCA immobilized on EP showed a
slightly higher Km value than the soluble enzyme.
Compared with free enzyme, a large fraction of the
immobilized enzyme locates in the pores of the
supports, which might decrease the enzyme-sub-
strate binding due to mass transfer limitations and
result in an increase in Km (Shibasaki-Kitakawa et al.
1998). However, the Km values of CCA immobilized
on EP-PEI and EP-EDA were even lower than that
of the free enzyme, indicating that a higher substrate
affinity was obtained after the immobilization on the
cationic supports. Among the three immobilized
enzymes, the EP-PEI immobilized enzyme had the
smallest Km value, followed by EP-EDA-CCA, with
the Km values reflecting the zeta potentials of the
corresponding supports and their enzyme derivatives.
In multiphase systems with immobilized enzymes,
biocatalysis depends on the mobility and partition of
substrates (Cantone et al. 2013). The zeta potential
of the substrate and enzyme preparations affect
the diffusion of substrate from the solution to the
microenvironment of immobilized CCA, and the
type and content of groups on the support are often
important for solute adsorption (Meder et al. 2013).
Therefore, the lower Km values for CCA immobi-
lized on the cationic supports (EP-PEI and EP-
EDA) than that of the free enzyme might be ascribed
to the increased localized concentration of the
negative charged substrate (CPC) near the immobi-
lized enzymes (Meder et al. 2013).
Kinetic parameters of the CCA preparations
The kinetic parameters of CPC hydrolysis were
determined for both the free and immobilized CCAs
(Table 2). The Lineweaver-Burk plot was linear,
indicating that the kinetics of the free and immobi-
lized CCA followed the Michaelis-Menten model.
100
80
60
40
20
0
The immobilized enzymes displayed lower Vmax
(maximal initial reaction velocity) and Vmax/Km
values in comparison to the free counterparts pos-
sibly due to enzyme conformational changes induced
by the support. EP-PEI-CCA had a larger Vmax than
the enzyme derivatives of EP-EDA and EP-CCA.
In addition, the content of amino groups on the
support might affect the solutes adsorption, leading
to an easier access of the substrate to the enzymes
on EP-PEI.
0
200
400
600
800
1000
NaCl concentration (mM)
Regeneration and reuse of EP-PEI
Figure 4. Adsorption–desorption of a protein from different ionic
Reuse from the supportability of the support is
an advantage of reversible immobilization, and it
requires the attached enzyme to be desorbed under
certain conditions. Increasing the ionic strength or
changing the pH of the solution weakens the inter-
actions between the protein and the ionic exchanger
(Torres et al. 2002). As shown in Figure 5, CCA was
almost completely desorbed from the EP-PEI by
using a high concentration of NaCl (e.g., 2 M) at low
supports. Symbols: (g) EP-EDA and ( ) EP-PEI. The residual
ꢁ
activity is expressed as a relative percentage of the original activity.
Table 2. Kinetic constants of free CCA and immobilized CCA.
EP-PEI-
CCA
EP-EDA-
CCA
EP-
CCA
Free
CCA
Km(mM)
Vmax (mmol/min/mg)
22
6.2
0.28
30
6.0
0.2
46
5.8
0.13
43
15
0.35
V
max/Km

8
Y. Wei et al.
especially for the enzyme immobilization on ionic
exchange supports.
100
80
60
40
20
Acknowledgements
We would like to thank Mr. D. Quiroga for his
language editing assistance in preparing the manu-
script. We thank Mr. W. Wei from Sunresin New
Materials Co. Ltd. for supplying the epoxy support
LX-1000EPC4. We thank Mr. Feng Yan from North
PR China Pharmaceutical Co. Ltd. for supplying the
sample of CPC and 7-ACA.
0
0
1
2
3
4
5
6
Cycles of support regeneration
Figure 5. Regeneration and reusability of the EP-PEI support.
The residual activity is expressed as a relative percentage of the
original activity.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content of the paper.
pH value (pH 3.0). The support could be reused for
several cycles to immobilize the enzyme with high
activity yield.
This work was supported by the National Science
Foundation of PR China (21276023; 21476025),
Beijing Natural Science foundation (2143041) and
the Fundamental Research Funds for the Central
Universities of PR China (FRF-BR-11-002A; FRF-
SD-12-007A).
For biocatalysts applied in industry, the costs of
immobilization depend heavily on the carrier, which
is often expensive (Cantone et al. 2013). Certainly,
the reusability of the EP-PEI support can make
biocatalytic process of CCA more cost-effective and
economically feasible.
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