An Approach for the Improved Immobilization of Penicillin G Acylase onto

Macroporous Poly(Glycidyl Methacrylate-co-Ethylene Glycol Dimethacrylate)

as a Potential Industrial Biocatalyst
 za, Sonja M. Jakovetic, and Andrea B. Stefanovic
Zorica D. Knezevic-Jugovic, Milena G. Zu
Dept. of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, University of Belgrade,
Karnegijeva 4, Serbia

Enis S. Dzunuzovic, Katarina B. Jeremic, and Slobodan M. Jovanovic

Dept. of Physical Chemistry and Electrochemistry, Faculty of Technology and Metallurgy, University of Belgrade,
Karnegijeva 4, Serbia

DOI 10.1002/btpr.2181
Published online October 21, 2015 in Wiley Online Library (wileyonlinelibrary.com)

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to
produce major advances in pharmaceutical processing industry and the enzyme stability
enhancement is still a significant challenge. The objective of this study was to improve cata-
lytic performance of the covalently immobilized PGA on a potential industrial carrier,
macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-co-
EGDMA)], by optimizing the copolymerization process and the enzyme attachment proce-
dure. This synthetic copolymer could be a very promising alternative for the development of
low-cost, easy-to-prepare, and stable biocatalyst compared to expensive commercially avail-
able epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA-co-
EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to
have higher activity yield compared to copolymerization in a cast. Optimal conditions for
the immobilization of PGA on poly(GMA-co-EGDMA) microbeads were 1 mg/mL of PGA in
0.75 mol/L phosphate buffer pH 6.0 at 258C for 24 h, leading to the active biocatalyst with
the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA
could contribute to the enhanced stability of the biocatalyst by inducing secondary interac-
tions between the enzyme and the carrier, ensuring multipoint attachment. The best balance
between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization
factor 22.2) was achieved for the partially modified PGA. V C 2015 American Institute of

Chemical Engineers Biotechnol. Prog., 32:4353, 2016

Keywords: penicillin G acylase, immobilization, poly(glycidyl methacrylate-co-ethylene
glycol dimethacrylate), suspension copolymerization, chemical amination, Eupergit C

Introduction ods for immobilization of PGA have already been described

Penicillin G acylase (PGA, E.C. 3.5.1.11) is an enzyme
mostly used in pharmaceutical industry in the production of
semisynthetic b-lactam antibiotics.1 PGA catalyzes deacyla-
tion of penicillin G (Pen G) to 6-aminopenicillanic acid (6-
APA), an intermediate for production of semisynthetic peni-
cillins such as amoxycillin and ampicillin.2 In order to
reduce the expense of producing 6-APA and semisynthetic
penicillins, the immobilized PGA with improved activity and
stability is preferred.3 Namely, the usage of the immobilized
PGA enables better operational stability and control of reac-
tions, more efficient recovery and reuse of the biocatalyst,
and application of different reactor designs.4 Various meth-
Contract grant sponsor: Ministry of Education, Science and Technologi-
cal Development of Serbia.
Additional Supporting Information may be found in the online ver-
sion of this article.
Correspondence concerning this article should be addressed to Z. D.
Knezevic-Jugovic at zknez@tmf.bg.ac.rs.
in literature including adsorption and covalent attachment to
porous organic5 and inorganic carriers,6,7 inclusion in and
attachment to biopolymer gels,8 and carrier-free techniques3;
among them, the covalent immobilization of PGA onto
epoxy-activated carriers has drawn considerable interest.9
Epoxy-carriers are very convenient for the multipoint
covalent attachment of enzymes considering that the present
epoxy groups could bind to amino, thiol, and hydroxyl
groups of the enzyme molecules.1012 Kinetic and thermo-
dynamic stabilization of the 3-D structure of the enzyme
appears to be achieved by the formation of the rigid
enzyme-carrier linkages. However, the random immobiliza-
tion may not promote any additional conformational stabili-
zation of the immobilized enzymes. In fact, the enzyme
activity and stability may be reduced during the immobiliza-
tion because immobilization conditions could cause the
enzyme denaturation or undesirable protein reorientation and
conformational changes.13 Many different experimental vari-
ables (immobilization conditions, density of reactive groups,

C 2015 American Institute of Chemical Engineers

V 43
44
and other characteristics of carriers, etc.) should be consid-
ered for the proper design of an immobilized PGA system
possessing improved stability and high activity retention.
The stabilization factor of PGA molecules has been shown
to be significantly increased by multipoint attachment of sev-
eral e-NH2 lysine groups of the protein surface to carriers
functionalized with aldehyde groups such as glyoxyl carriers14
or to epoxy groups of some commercial epoxy carriers such
as Eupergit or Sepabeads.3 However, commercial epoxy car-
riers are not satisfactory since they are too expensive for com-
mon use. Moreover, epoxy groups can be introduced on other
carriers by glycidyl methacrylate as the component of an acry-
late polymer.15 Although the multipoint attachment has been
generally accepted for the enzyme immobilization on these
carriers, if the target enzyme is poor in Lys groups, the prop-
erties of the immobilized enzyme may be adverse. Likewise,
better possibilities of yielding a more intense multipoint cova-
lent attachment could be achieved by chemical modification
of enzymes resulting in an enzyme surface enriched in
reactive groups. For example, Rodrigues et al. studied the
immobilization of aminated lipase on glyoxyl agarose and
determined that the chemical amination of lipase after immo-
bilization on glyoxyl-agarose beads improved the multipoint
covalent attachment of this enzyme.16 Furthermore, the immo-
bilization of the chemically modified PGA and glutaryl acy-
lase on glyoxyl carrier has been shown to increase the
enzyme stability (by a fourfold factor in the case of PGA)
compared to the stability of the immobilized but nonmodified
enzyme.14 In spite of these advantageous studies on PGA
immobilization, further development of new techniques for
PGA immobilization on chemically stable, inexpensive, and
industrially applicable carriers is still necessary to improve the
catalytic performance of the immobilized enzyme and to
reduce the cost of process.
The aim of this work was to improve the performance of
PGA from Escherichia coli for the penicillin G hydrolysis by
the means of covalent immobilization on the highly reactive
copolymer synthesized in our laboratory. The macroporous
copolymers of glycidyl methacrylate (GMA) and ethylene
glycol dimethacrylate (EGDMA) [abbreviated poly(GMA-co-
EGDMA)] could be very promising for the development of
low-cost, easy-to-prepare, and stable biocatalysts, providing
suitable surface for covalent binding of enzymes and suitable
microenvironment. Furthermore, it exhibits some interesting
properties such as excellent thermal and chemical stability
and form versatility (powder, microbeads, plate, fibers, rods,
and membranes) making it very useful as matrix for column
packing in different types of enzyme reactors.
This article discusses the different copolymerization and
immobilization procedures to enhance the properties of PGA
immobilized on poly(GMA-co-EGDMA). Considering that
porosity and geometry of the carrier is very important for
immobilization of the enzyme, the influence of type of
copolymerization reaction and conditions on the activity and
stability of immobilized PGA was examined. For this pur-
pose, the poly(GMA-co-EGDMA) was obtained in three dif-
ferent ways: in the shape of microbeads by suspension
polymerization, and in the shape of rod and plate by copoly-
merization in a cast and used as carriers for PGA immobili-
zation. Similarly, the immobilization parameters for PGA
immobilization on poly(GMA-co-EGDMA) by the conven-
tional method based on direct enzyme binding on polymers
via epoxy groups were optimized (pH, ionic strength, tem-
Biotechnol. Prog., 2016, Vol. 32, No. 1
perature, duration of immobilization, and initial concentra-
tion of PGA in attachment solution).
To further enhance the stability of the immobilized PGA,
an attempt of variation of this covalent method has been
examined. The concept involved the introduction of addi-
tional amino groups into enzyme molecules allowing the
completion of the immobilization process, i.e., the formation
of multiple cooperative chemical bonds. In order to evaluate
effectiveness of the obtained immobilized system, the activ-
ity and thermal stability of the produced biocatalyst were
compared to those obtained with nonmodified PGA and by
the immobilization of PGA on the commercial epoxy carrier
such as Eupergit C.
Materials and Methods
Materials
PGA from E. coli was kindly donated by DSM Anti-
Infectives (Delft, The Netherlands). Polyvinylpyrrolidone
(PVP) (Kolidon 90, molar mass 360,000), GMA, EGDMA,
1-ethyl-3-(dimethylamino-propyl) carbodiimide (EDAC),
cyclohexanol, decanol, tetradecanol, and hexadecanol were
purchased from Sigma-Aldrich (St. Louis, MO, USA). p-
Dimethyl aminobenzaldehyde (PDAB) used for determina-
tion of the PGA activity was also purchased from Sigma-
Aldrich (St. Louis, MO, USA). Eupergit C was a kind gift
from Rohm GmbH & Co., Degussa (Darmstadt, Germany).
All other chemicals were of analytical grade products and
were purchased from Merck AG (Darmstadt, Germany).
Methods
Preparation of the Carrier. Suspension polymeriza-
tion. The polymer in microbeads form was prepared by
suspension copolymerization in a 1000 mL mechanically
stirred batch reactor with temperature and pH control. The
typical copolymerization conditions were as follows: 240 g
of a 1% aqueous solution of PVP was mixed with monomer
phase that contained monomers (24.2 g of GMA and 10.3 g
of EGDMA), 0.8 g of initiator azo-bis-isobytironitrile
(AIBN), and inert phase (36.5 g of cyclohexanol and 9.1 g
of aliphatic alcohol: decanol or tetradecanol or hexadecanol).
The mixture was stirred for 2 h at 708C and then for 6 h at
808C, as previously described.17,18 After completion of the
reaction, the copolymer particles were separated, washed
with water and ethanol, kept in ethanol overnight, and then
dried in a vacuum oven at 408C. The microbeads fraction
with diameter in the range of 150500 lm was used for fur-
ther investigation.
Polymerization in a cast. The monolithic copolymer
samples in the shape of plate and rod were obtained by
copolymerization in the cast. The copolymer plates were
obtained by copolymerization of the monomer phase in the
presence of inert component between two glass plates sepa-
rated with 2-mm-thick Teflon spacer, while the rod-shaped
copolymer samples were obtained by polymerization of the
same reaction mixture conducted in a glass tube with an
inner diameter of 1 cm and a length of 5 cm. All other con-
ditions were the same as for suspension copolymerization.
Typically, the basic form of the copolymer was synthesized
by the addition of tetradecanol as a porogen. The ratio of
inert phase to monomer phase was 1.3 for both types of
copolymerization reaction (suspension and copolymerization
Biotechnol. Prog., 2016, Vol. 32, No. 1
in a cast). To extract the inert component, the obtained plate
and rod copolymer samples were kept in ethanol for 48 h,
and then dried in a vacuum oven at 408C.
Characterization of the Carrier and Immobilized
Enzyme. The granulometric composition of macroporous
copolymers obtained by suspension copolymerization was
determined by sieve analysis. Determination of the pore size
distribution was carried out on mercury porosimeter (Carlo
Erba 2000). The morphology of the lyophilized polymers
poly(GMA-co- EGDMA) and Eupergit C samples were ana-
lyzed using Scanning Electron Microscope (JEOL JSM5800
SEM) with a resolution operated at 30 kV and magnification
of 20003.
The chemical characterization of poly(GMA-co EGDMA),
Eupergit C, and PGA immobilized onto the carriers has been
done by Fourier-transform infrared (FT-IR) spectroscopy
using a Bomem MB 100 (Hartmann & Braun) FT-IR Spec-
trophotometer. Samples in an amount of 10 wt% were mixed
and ground with 100 wt% of potassium bromide and then
compressed into a pellet under a pressure of 11 t, for about
1 min, using a Graseby Specac Model: 15.011 press. The
Spectra were recorded in the 4004000 cm21 wave number
range, at 258C and at 4 cm21 spectral resolution.
The concentration of epoxy groups was determined by dis-
persing the copolymer sample in the 0.1 M solution of tetra-
ethylammonium bromide in acetic acid and its titration with
0.1 M perchloric acids solution with crystal violet as an
indicator.
One-Step Immobilization of PGA via Epoxy Groups
(Method I). First, the conventional method for enzyme
immobilization on epoxy carriers was applied based on
direct enzyme binding on the polymer via epoxy groups in
one step.19 The enzyme solution (128 U) in different buffers
(10 mL, pH 5.09.0) was incubated for predetermined time
in the presence of the synthesized copolymer (250 mg, dry
state) under stirring (120 rpm). The buffers used were 1 mol/
L acetate buffer (pH 5.0; 5.5), phosphate buffer (pH 6.0;
6.5), and TRIS buffer (8.0; 9.0). The other immobilization
conditions were also optimized, changing the ionic strength
of predetermined buffer (0.51.5 mol/L), temperature (5
358C), initial enzyme concentration (from 0.14 to 2.24 mg/
mL), and immobilization time (896 h). After that, the
immobilized enzyme was separated by vacuum filtration,
washed several times with correspondent buffer, and filtered
again to obtain it in a wet form. The amount of protein was
followed using the standard Lowry assay.20 The amount of
the bound enzyme was determined by the difference between
the initial amount of protein in the initial immobilization
solution and the amount remaining in the filtrate and wash-
ing solutions.
For comparison purposes, PGA was also immobilized on
commercial Eupergit C carrier using the same conventional
method under the specified conditions. Namely, dry polymer
(250 mg) was added into 10 mL of native PGA solution in
1.25 mol/L potassium phosphate buffer pH 8.0 (128 U of
enzyme). The immobilization was carried out for 48 h at
258C under mild stirring (120 rpm). The produced prepara-
tion was filtered, washed with 1.25 mol/L potassium phos-
phate buffer pH 8.0, and stored at 48C prior to use.
Immobilization of PGA Followed by Subsequent Amination
(Method II). The immobilization procedure consisted of
two main steps: (1) immobilization of PGA on the copoly-
mer beads via epoxide group and (2) amination of the immo-
45
bilized enzyme. The amination was carried out as follows:
1 g of PGA immobilized onto poly(GMA-co-EGDMA)
(pPGA) or onto Eupergit C (ePGA) was added to 49 mL of
1 mol/L ethylenediamine at pH 4.75. Different amounts of
solid EDAC were added to the suspension to a final concen-
tration of 1022 or 1023 mol/L. After 90 min of gentle stir-
ring at 258C, the immobilized preparations were filtered and
incubated for 4 h in 0.1 mol/L hydroxylamine solution at pH
7 to recover the modified tyrosines.16 The immobilized prep-
arations were filtered, thoroughly washed with the buffer,
and named as pPGA 10 mM and ePGA 10 mM (immobi-
lized PGA modified with 1022 mol/L EDAC and highly
modified immobilized PGA); pPGA 5 mM and ePGA 5 mM
(immobilized PGA modified with 5 3 1023 mol/L EDAC-
partially modified immobilized PGA); and pPGA 1 mM and
ePGA 1 mM (immobilized PGA modified with 1023 mol/L
EDAC-slightly modified immobilized PGA).
Enzyme Activity Assay. Reaction mixture used for deter-
mination of PGA activity (3.5 mL) consisted of penicillin G
sodium salt (0.04 mol/L in 0.1 mol/L phosphate buffer at pH
7.92), 0.1 g of immobilized PGA or 0.5 mL of free PGA
solution in the correspondent buffer. Reaction was carried in
an orbital shaker set at 378C and 100 rpm for 3 min, after
which 2.5 mL of ethanol was added to inhibit further reac-
tion. The amount of enzymatic product, 6-APA, was fol-
lowed spectrophotometrically at 415 nm using PDAB as
indicator, as previously described elsewhere.21 One unit of
PGA activity was defined as the amount of free or immobi-
lized enzyme required to produce 1 mmol of 6-APA per
minute under the assay conditions. Enzyme coupling yield
was calculated using the following equation:
YE %5
P1
3100 (1)
P0
where P1 represented the amount of the immobilized protein
and P0 was the initial amount of protein in the enzyme cou-
pling solution determined by Lowry method.
Activity coupling yield was also used for evaluation of
immobilization efficiency and it was calculated as follows:
SA2
YA %5 3100 (2)
SA1
where SA1 and SA2 refer to the specific activities of free and
immobilized PGA, respectively.
Thermal Stability Assays. The thermal stability assays
were performed at 508C in 0.1 mol/L phosphate buffer, pH
7.92 with 0.1 g of the biocatalysts. In a predetermined time
interval, a sample was removed and assayed for enzymatic
activity as described above.
The first-order enzyme deactivation model with residual
activity was used to describe the experimental thermal deac-
tivation data for biocatalysts:
A5A0 e2kd t 1A1 (3)
where A0 and A are the initial and residual enzyme activity
at time, respectively, A1 is the residual enzyme activity, and
kd is the first-order deactivation rate constant. Results
obtained in the thermal stability study were fitted with the
first-order kinetic model using Matlab software. The constant
kd, biocatalysts half-time t1/2, and stabilization factor F were
used to evaluate obtained results. Stabilization factor is
46
Figure 1. SEM micrograph of the cross-section of poly(GMA-co-EGDMA) at scale bar (a) 5 and (c) 10 lm and the cross-section of
Eupergit C at scale bar (b) 5 and (d) 10 lm.
calculated by dividing half-time of the obtained biocatalyst
with half-time of free PGA.
All data are the averages of triplicate samples and were
reproducible within 65% of accuracy. Mean and standard
deviation of the results from at least three independent
experiments were calculated using Microsoft Excel software.
Results and Discussion
Polymer and biocatalyst characterization using scanning
electron microscopy (SEM) and Fourier-transform infrared
(FT-IR) spectroscopy
The copolymer poly(GMA-co-EGDMA) was prepared from
GMA and EGDMA (70:30 mass%) under different copoly-
merization conditions and used for PGA immobilization.
It was previously reported that increase in EGDMA con-
centration in the monomer mixture from 10 to 40 mass%
significantly reduced the size of the pores, with a concurrent
increase in the porosity, pore volume, and the specific sur-
face area. Nevertheless, this phenomenon continued up to
EGDMA concentration of 45 mass%, after which further
increase in EGDMA concentration did not cause significant
increase in the specific surface area.22
Typically, the basic form of the copolymer was synthesized
by the addition of tetradecanol as a porogen, except when the
influence of the type of aliphatic alcohol (decanol, tetradeca-
nol, and hexadecanol) in an inert component of suspension
polymerization on immobilization of PGA was examined.
Some properties of basic form of the copolymer in the shape
of spherical microbeads obtained by suspension copolymeriza-
tion have been described earlier: specific surface area deter-
mined by BET method from the low-temperature N2
adsorption isotherms obtained at 77 K seemed to be 27.6 m2/g.
Biotechnol. Prog., 2016, Vol. 32, No. 1
On the other hand, the specific surface area obtained from
cumulative pore volume distribution curves has been found to
be 26 m2/g, pore volume 1.040 mL/g, pore diameter that corre-
sponds to the half of pore volume was 270 nm, and average
pore diameter 151 nm.18 Also, it appeared that the type of
copolymerization had a significant influence on the porosity
parameters of the obtained copolymer. Namely, copolymer
samples in the shape of plate and rod obtained by copolymer-
ization in the cast had pore diameters larger than 400 nm (407
and 420 nm, respectively) not achieved by suspension co-
polymerization. This is likely due to the partial miscibility of
monomer, inert component, and water in suspension copoly-
merization and the influence of this on the separation of the
new phase during the reaction.18
The average pore diameter of poly(GMA-co-EGDMA)
microbeads (151 nm) appeared to be about 15 times larger
than that for Eupergit C (10 nm).23 In this work, we present
some other properties of this material, which are of importance
for enzyme immobilization as well as their comparison to the
properties of the commercial epoxy carrier such as Eupergit C.
The morphology of the lyophilized polymers poly(GMA-co-
EGDMA) and Eupergit C was analyzed using Scanning Elec-
tron Microscope. The obtained scanning electron microscopy
(SEM) micrographs are presented in Figure 1.
The morphological appearance of the particles of synthe-
sized copolymer, showed in Figure 1a,c clearly indicates
porous structure of the copolymer, where each particle of the
copolymer consists of interconnected microspheres. On the
other hand, Figure 1b,d demonstrates the smooth surface of
Eupergit C.
In the further work, the PGAcopolymer conjugate
(pPGA) was characterized, and the mechanism of interaction
between the polymer and the protein was studied using FT-
IR. The FT-IR spectra of the poly(GMA-co-EGDMA), PGA
Biotechnol. Prog., 2016, Vol. 32, No. 1
Figure 2. A comparative FT-IR spectra of (a) poly(GMA-co EGDMA) (curve 1) and pPGA (curve 2) and (b) Eupergit C (curve 1)
and ePGA (curve 2).
immobilized on poly(GMA-co-EGDMA) pPGA, Eupergit C,
and PGA immobilized on Eupergit C (ePGA) are presented
in Figure 2.
The FT-IR spectrum of both copolymers, poly(GMA-co
EGDMA) and Eupergit C, exhibited characteristic bands at
848 and 908 cm21 due to epoxy ring vibrations (cCAO
epoxy) and around 1260 cm21 as a consequence of d(CAH)
epoxy. The presence of epoxy groups in IR spectra was also
proved for both polymers from the presence of strong bands
at around 3,000 cm21 (mCAH epoxy) (2,989 and 2,998 cm21
for Eupergit C and poly(GMA-co EGDMA), respectively).
Methylene vibration was observed between 2954 and
2995 cm21. Antisymmetric and symmetric stretching bands
of carbonyl groups were observed at 1384 and 1257 cm21
for Eupergit C and ePGA, and at 1390 and 1260 cm21 for
poly(GMA-co EGDMA) and pPGA, respectively. The low
intensity of the band at 908 cm21, characteristic for epoxy
groups, in Eupergit C FT-IR spectrum, indicated that the pol-
y(GMA-co EGDMA) contained higher concentration of
functional groups. Concentration of epoxy groups available
for the reaction with the enzyme could be different from
those obtained by FT-IR analysis since only the groups
located on the immediate surface of microbeads can actually
interact with PGA. Determined values of epoxy group con-
centration on the surface of synthesized copolymer microbe-
ads and commercial epoxy-carrier, Eupergit C, were 2.16
47
and 0.42 mmol/g, respectively, which was in accordance
with the results obtained by FT-IR analysis. This is also con-
sistent with previous reports for poly(GMA-co-EGDMA) and
Eupergit C by other authors.18,23
It seemed that some major chemical changes occurred due
to the PGA immobilization onto the polymer. The most
obvious distinguishing features were that the immobilized
PGA spectra contained an intense broad band in the range of
32003600 cm21 due to NAH stretching and OAH stretch-
ing vibrations of the enzyme amino and OH groups. The dis-
appearance of the bands at 3,000 and 908 cm21 indicated
the opening of epoxy rings. On the other hand, the appear-
ance of the band at 1,109 cm21 was characteristic for CAN
stretching vibrations, suggesting that epoxy groups were
altered due to the reaction with enzyme amino groups.
Optimization of the preparation of the carrier and
conditions for PGA immobilization onto poly(GMA-co-
EGDMA)
The effects of pH of buffer solution in the range of 5.0
9.0 on the enzyme and activity coupling yields for PGA
immobilization on the poly(GMA-co-EGDMA) samples
were studied. The samples that were in the shape of
microbeads and in the shape of plate, 2 mm tick, or a rod
were synthesized by suspension copolymerization and
48
Figure 3. (a) Enzyme coupling yield and (b) activity yield as a function of pH of the buffer used for the PGA immobilization on poly
(GMA-co-EGDMA) synthesized by suspension polymerization and polymerization in a cast in three different forms (shapes).
copolymerization in the cast, respectively. The results are
shown in Figure 3.
The activity of the pPGA in all three cases clearly depended
on the pH of the buffer used for the immobilization. The
enzyme exhibited good binding between pH 6.0 and 6.5, but
the activity decreased rapidly for enzyme immobilized at
higher pH. Optimum binding was achieved with 1.0 mol/L
phosphate buffer at pH 6. Under this condition, about 463.9,
445.9, and 424.4 U were immobilized per gram of dry carrier
with enzyme coupling yield of 90.6%, 87.1%, and 82.9% and
activity yield of 56.9%, 16.2%, and 19.7%, for carrier in the
shape of microbeads, and in the shape of plate and rod, respec-
tively. It can be concluded that the enzyme denaturation during
the immobilization was very low considering that pH depend-
ency of activity yield followed pH dependency of enzyme cou-
pling yield. Similarly, the carrier synthesized by suspension
copolymerization apparently was much better for the immobili-
zation of PGA than the other two forms, as its activity yield
was the highest. The results indicated that for the immobiliza-
tion of the enzyme, the most important was interfaces between
the carrier and the enzyme solution, considering that the car-
riers synthesized in the cast had higher porosity but lower
interfaces than the carrier synthesized by suspension copoly-
merization. Theoretically, the copolymer synthesized in a cast
could present different surfaces for enzyme binding, perhaps
offering some advantages for specific enzymes. They should
be investigated further particularly with respect to the method
for obtaining porous surfaces. However, in this case, the
copolymerization in the cast did not improve the copolymer
performances for PGA immobilization. Therefore, the carrier
synthesized by suspension polymerization was used in all sub-
sequent experiments.
The influence of the type of aliphatic alcohol (decanol, tet-
radecanol, and hexadecanol) in an inert component during
the suspension copolymerization on PGA immobilization
was further investigated. It was apparent that a decrease in
carbon atoms in the aliphatic alcohol at the same concentra-
tion of alcohol in the inert component led to decrease in
pore radius, and consequently increase in specific surface
area (Supporting Information, Table SI). However, this
increase in specific surface area did not cause expected
increase in achieved activity yields, and the highest activity
yield of 57.7% was obtained when hexadecanol was used as
an inert component. Similar values were obtained with tetra-
Biotechnol. Prog., 2016, Vol. 32, No. 1
decanol and decanol (Supporting Information, Table SI). The
enzyme coupling yields were also similar for all aliphatic
alcohols used (90.5% 6 2.20), revealing that the type of ali-
phatic alcohol in the inert component of suspension copoly-
merization did not significantly influence the immobilization
of PGA in this case.
Optimization of other conditions of PGA immobilization
onto poly(GMA-co-EGDMA) microbeads
Optimization of PGA immobilization on poly(GMA-co-
EGDMA) was continued by investigation of the effects of
different ionic strengths (0.51.5 mol/L) on the enzyme cou-
pling yield and activity yield at the previously determined
optimal pH. Immobilization of protein on epoxy carriers is
usually conducted at high ionic strengths as it reduces
adsorption based on electrostatic interactions and promotes
enzyme adsorption via hydrophobic residues, which is fol-
lowed by slow formation of covalent bond between carrier
epoxy groups and enzyme by a ring-opening reaction
(Scheme 1a).
The results are shown in Figure 4a. The highest enzyme
coupling yield of 92.6 6 0.56%, correspondent to the activity
of around 474.4 U per gram of dry carrier was achieved at
buffer concentration of 1 mol/L, but correspondent activity
yield of 54.2 6 1.46% was less by 30% than the one obtained
when buffer concentration of 0.75 mol/L was used, indicating
an inadequate enzyme binding to the carrier and more intense
denaturation at higher concentrations. Hence, buffer concen-
tration of 0.75 mol/L was used for further studies.
Subsequent analysis included finding of the optimal tem-
perature for PGA immobilization, hence immobilization was
carried out at different temperatures, in the range of 5358C
in previously determined optimal buffer (0.75 mol/L phos-
phate buffer, pH 6.0). The results are shown in Figure 4b,
and they indicate that immobilization temperature had no
significant effect on the enzyme coupling yield
(66.5 6 1.15%). However, the activity yield had an apparent
maximum at 258C. The results revealed that PGA retained
its active conformation after immobilization on poly(GMA-
co EGDMA) conducted at 258C. Therefore, further experi-
ments were exclusively carried out at 258C.
Further optimization of PGA immobilization on the poly(-
GMA-co EGDMA) consisted of finding the optimal
Biotechnol. Prog., 2016, Vol. 32, No. 1 49

Sch 1. Schematic representation of (a) conventional method for PGA immobilization on epoxy carriers based on direct enzyme bind-
ing on the polymer via epoxide groups in one step and (b) modification of the procedure including post-treatment consisting of
the chemical amination of PGA with ethylenediamine (after activation of the carboxylic function with carbodiimide).

Figure 4. The effect of (a) ionic strength and (b) temperature on enzyme coupling yield and activity yield.

immobilization time and initial PGA concentration in the
coupling solution, and the obtained results are shown in Fig-
ure 5ac, respectively. It appeared that immobilization was
complete within 24 h. Enzyme coupling yield remained
almost constant with further increase in immobilization time
indicating that available enzymes reacted with the carrier in
the first 24 h, hence making prolonged immobilization time
unnecessarily. The decrease in the activity yield that ensued
after 24 h may be attributed to the denaturation of PGA. The
highest enzyme coupling yield of 66.7 6 2.18% and the
highest activity yield of 70.3 6 2.85% were obtained after
24 h immobilization; therefore, 24 h was set as optimal
immobilization time.
The initial concentration of PGA in the coupling solution is
another important factor influencing the catalytic performance
of the immobilized PGA. This influence was depicted through
the values of both enzyme coupling yield (Figure 5c) and
activity yield (Figure 5b). It was observed that the enzyme
coupling yield increased with increasing initial PGA concen-
tration up to 72.3 6 5.65%, and then reached saturation at the
50
Figure 5. The influence of (a) immobilization time on enzyme
and activity coupling yield and the influence of ini-
tial PGA concentration on the (b) mass of enzyme
bound and activity yield and (c) enzyme coupling
yield.
PGA concentration of around 1 mg/mL. Concurrently, the
enzyme loading on the copolymer increased almost linearly
by increasing PGA initial concentration and the highest
enzyme loading was obtained for the initial concentration of
2.24 mg/mL. It was possible to immobilize as much as
59.3 6 1.52 mg protein/g dry weight of the copolymer,
revealing a rather high affinity of the copolymer toward the
PGA molecules. This loading capacity is higher than, or com-
Biotechnol. Prog., 2016, Vol. 32, No. 1
parable with that achieved using other carriers such as porous
magnetic poly(GMA-MBAA-NVP) composite microspheres
containing epoxy groups24 or carrier specifically design in an
effort to improve the loading amount of PGA, increasing the
interaction between PGA and the carrier such as chitosan
grafted coreshell porous glass beads.25 It was estimated
roughly that the maximum loading of PGA on the poly(GMA-
co EGDMA) microbeads can reach 186.15 g per gram of poly-
mer (6.74 g/m2), assuming that each protein molecule was
linked by one epoxy functional group and taking into account
the density of epoxy functional groups on the poly(GMA-co
EGDMA) beads (2.16 mmol/g). Considering this high epoxy
group density, a large fraction of immobilized PGA molecules
was apparently chemically linked to the carrier.
On the contrary, the highest activity yield of 89.2 6 2.44%
was achieved for the lowest initial concentration of 0.14 mg/
mL, likely due to the close packing of PGA on poly(GMA-co
EGDMA) surface at higher enzyme loading resulting in the
mass transfer limitations. However, when discussing the indus-
trial relevance of an immobilized enzyme, its activity per gram
of carrier is also of importance. The main challenge with
enzyme immobilization is to obtain biocatalyst with high reten-
tion of catalytic activity possessing a satisfactory amount of
protein bound. When the activity per gram of carrier was ana-
lyzed, it seemed that a bell-shaped curve was obtained with a
maximum activity of 252.7 6 10.04 U/g dry microbeads at a
loading of around 25.6 6 2.52 mg/g (Supporting Information,
Figure SI). Above the maximum, the activity of the immobi-
lized enzyme dropped, possibly due to the mass transfer prob-
lems associated with diffusion of substrate and product into the
carrier particles and access to the enzymes active site. There-
fore, the initial concentration of PGA of 1 mg/mL in attach-
ment solution was set as optimal and used for further studies.
The results are similar to the results of previous studies con-
cerning immobilization of PGA on silica nanoparticles, where
the specific activity determined by a kinetic assay decreased by
around 60%, when increasing the enzyme loading from 14.9 to
55.8 mg/g.26 Because of the fact that no major changes in the
secondary structure have been found, diffusion limitations
appeared to be the main reason for the decline of the activity.26
However, besides being highly efficient, a carrier used for
industrial applications must also be produced at affordable
costs. The copolymer is normally prepared for research pur-
poses on a small scale, in a research lab, and costs could not
be explicitly considered. A rough cost analysis based on pri-
ces of chemical required for synthesis of 10 g of the copoly-
mer (21.9 e, Catalog prices, Sigma Aldrich) revealed that
the poly(GMA-co-EGDMA) synthesized in our laboratory is
many times cheaper than the commercially epoxy polymers
(Eupergit C or Sepabeads). However, in this analysis, we did
not consider additional costs including labor cost, labor
requirements, equipment cost, and others. Comparing to sev-
eral covalent binding protocols reported in the literature, the
poly(GMA-co EGDMA) copolymer is preferred to use as a
carrier because of its low cost, approved use in the pharma-
ceutical industry, easy production, and efficient removal
from the reaction medium for reuse.
PGA immobilization followed by subsequent amination
(method II)
The effects of the chemical amination of the enzyme after
the immobilization procedure were studied in order to
improve the activity and stability of PGA immobilized on
Biotechnol. Prog., 2016, Vol. 32, No. 1
Figure 6. Distribution of Lys, Asp, and Glu residues in PGA molecule. Lys is shown in yellow; Asp and Glu are shown in blue and
red. PGA is shown in (a) surface and (b) cartoon representation. 3-D structure was obtained from Protein Data Bank 1ai4
using Pymol.
Table 1. Results of the Immobilization of PGA from E. coli on Poly(GMA-co-EGDMA) and Eupergit C
Biocatalyst AIE (U/g dry beads)
pPGA 252.7
pPGA 1 mM 196.2
pPGA 5 mM 180.0
pPGA 10 mM 43.8
ePGA 211.0
ePGA 1 mM 163.7
ePGA 5 mM 90.9
ePGA 10 mM 36.7
AIE, activity of the immobilized PGA per gram of dry carrier; mE, mass of protein bound per gram of dry carrier; SA2, specific activity of free enzyme
is 13.65 U/mg.
poly(GMA-co-EGDMA) microbeads obtained by suspension
polymerization. For a comparison purpose, a control experi-
ment with commercial epoxy carrier such as Eupergit C has
also been performed. Chemical amination of the biocatalyst
seemed to be a very simple way to enrich the enzyme sur-
face in primary amino groups, making the enzyme more
reactive with the epoxy carrier at milder condition. The sur-
face carboxylic groups (Asp and Glu) were activated with
EDAC in the presence of ethylenediamine (Scheme 1b). The
reason for choosing these residues to be modified in PGA
was that they are numerous, easily reached, and easily modi-
fied with ethylenediamine (Figure 6). In the reaction, an
amide bond between the activated carboxylic groups of the
enzyme and one of the amino groups of ethylenediamine
formed, leaving one free primary amino group (Scheme 1b).
These introduced amino groups (pK value of around 9.2)14,27
appeared to be more reactive than the e-amino groups of the
lysyl residues of the protein surface.
Thus, after immobilizing PGA on poly(GMA-co-EGDMA)
or Eupergit C, it was possible, instead of blocking the
remainder epoxide groups to prevent any further reaction
between the enzyme and the carrier, to modify the already
immobilized enzyme by introducing more amino groups with
a high activity, and in this way to stimulate multipoint
immobilization. Three different PGA immobilized prepara-
tions were prepared by controlling the intensity of the multi-
interaction process between amino groups of PGA and
epoxide groups of the carriers and activity of biocatalysts
and amount of enzyme bound have been determined. The
immobilization results are summarized in Table 1.
51
mE (mg/g dry beads) SA1 (U/mg protein) YA (%)
25.6 9.87 72.3
25.6 7.66 56.14
25.6 7.03 51.55
25.6 1.71 12.53
23.6 8.94 65.5
23.6 6.93 50.8
23.6 3.85 28.2
23.6 1.55 11.4
Specific activities and activity yields of pPGA and ePGA
were 252.7 and 211.0 U/g dry beads and 72.3% and 65.5%,
respectively. It can be concluded that pPGA was more active
than ePGA, this was probably due to more porous surface
properties of the poly(GMA-co-EGDMA) microbeads reduc-
ing diffusion limitations of the substrate and products.
Chemical amination resulted in 27.788.6% activity loss, but
the effect was moderated by a reduction in the degree of
modification (amination). Thus, the highest loss of activity
was observed for the biocatalysts modified with the highest
amount of EDAC (pPGA 10 mM and ePGA 10 mM) while
the partially modified pPGA (pPGA 1 mM) retained a rather
high catalytic activity of 56.14%.
The immobilized enzyme stability often has a crucial role
in determining whether or not a system can be applicable for
industrial purposes. Study of thermal deactivation kinetics
at 508C, presented in Figure 7, revealed that the partially
modified pPGA 5 mM and totally modified pPGA 10 mM
produced an appreciable stabilization of the biocatalyst,
changing its thermal deactivation profile. By comparison of
the t1/2 values, presented in Table 2, it can be concluded that
the partially modified pPGA 5 mM and the highly modified
pPGA 10 mM were 1.5- and 3.7-fold more stable than con-
ventionally immobilized one and 22.2- and 53.7-fold than
free PGA, respectively. The stabilization factor was much
higher or consistent with values reported for PGA covalently
immobilized on different agarose derivatives,28 chitosan,8 or
heterofunctional thiol-reactive disulfide/epoxy supports29 but
lower than reported for the genetically modified PGA
which was covalently immobilized on Eupergit C.9 Namely,
52 Biotechnol. Prog., 2016, Vol. 32, No. 1

Figure 7. Thermal stability of (a) free PGA and different derivatives of pPGA: (b) free PGA and different derivatives of ePGA.

Table 2. Values of the Kinetic Constants Obtained with the First- immobilized PGA and 2.6- and 12.8-fold more stable than
Order One-Step Kinetic Model with Residual Activity for Free PGA free PGA, respectively. Highly modified ePGA 10 mM was
and the Immobilized Derivatives at 508C 12.3- and 23.9-fold more stable than conventionally immobi-
Biocatalyst kd (1/h) t1/2 F A1 lized PGA on Eupergit C and free PGA, respectively. The
Free PGA 2.34 0.30 1 0.15 results are in agreement with the fact that the stability of
pPGA 0.20 4.36 14.5 15.33 the covalently immobilized enzymes was closely related to
pPGA 1 mM 0.88 1.35 4.5 9.74
the number of bonds between the enzyme and carrier.30 An
pPGA 5 mM 0.122 6.67 22.2 15.43
pPGA 10 mM 0.059 16.10 53.7 19.36 enhancement of multiple-point attachment of enzyme to car-
ePGA 1.554 0.582 1.94 15.96 rier often led to an increase of enzyme stability making the
ePGA 1 mM 1.066 0.793 2.6 12.12 unfolding of the peptide chains more difficult. This result
ePGA 5 mM 0.201 3.848 12.8 6.41 was consistent with other rigid enzyme immobilizations
ePGA 10 mM 0.136 7.18 23.9 21.79
which often resulted in a rather high t1/2 values.31 However,
kd is the first-order deactivation rate constant; t1/2 is the half-life of it appeared that the number of bonds between enzyme and
biocatalysts; F is the stabilization factor; A1 is the residual activity. carrier necessary to achieve maximum stabilization depended
on enzyme and carrier. In addition, the activity was reversi-
Montes et al. obtained that the aminated PGA immobilized bly proportional to the number of bonds, making a design of
on glyoxyl agarose presented a half-life around two times an efficient enzyme immobilized system possessing high
higher than the unmodified immobilized PGA.28 Adriano activity retention and improved stability still a significant
et al. reported that the PGA immobilized on glutaraldehyde- challenge. In spite of the excellent thermal stability results,
activated chitosan by multipoint covalent attachment was pPGA 10 mM showed a rather low specific activity of only
4.9-fold more stable than the free enzyme at 508C, while the 43.8 U/g dry beads. The best balance between the activity
conventionally immobilized PGA was 2.7-fold more stable.8 yield (51.5%), enzyme loading (25.6 mg/g), and stability
Grazu reported stabilization of PGA with genetically intro- (stabilization factor 22.23) was achieved for the partially
duced cysteyl residues by multipoint covalent attachment on modified pPGA 5 mM.
heterofunctional carriers containing thiol-reactive disulfides
and epoxide groups (HDE supports).29 However, the immo-
bilization of this genetically modified PGA variant with a
Conclusions
unique cysteine at position 380 surrounded by 4 extra lysine The poly(GMA-co-EGDMA) synthesized in the shape of
residues (PGA-4Kb380) on Eupergit C by multipoint attach- microbeads by suspension copolymerization seemed to have
ment improved the enzyme stability 1500-fold in comparison much better performance for the immobilization of PGA
with the soluble PGA-4Kb380.9 Regarding that the soluble compared to the carriers in the shape of rod or plate synthe-
PGA-4Kb380 presented 28-fold lower thermal-stability than sized by copolymerization in a cast. The optimal condition
the wild-type PGA, the immobilization on Eupergit C of the for PGA immobilization on poly(GMA-co-EGDMA) were
lysine-enriched PGA promoted a 10-fold higher stabilization 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at
factor against temperature (558C). However, the obtained 258C for 24 h. This protocol led to the active immobilized
values of stabilization factor and half-life of biocatalysts biocatalyst for the Penicillin G hydrolysis. It seemed that
could not be directly compared with a majority reported in using identical enzyme concentrations, the direct enzyme
the literature for free/or immobilized PGA because of differ- binding on the poly(GMA-co-EGDMA) microbeads via
ent protocols for the thermal stability study used (different epoxy groups in one step resulted in an activity yield of
PGA variants, temperatures, presence of organic solvents, 72.3% at enzyme loading of 25.6 mg/g, while that for PGA
and others). immobilization on Eupergit was 65.5%. However, the immo-
In the case of PGA immobilization on Eupergit C in this bilized PGA on both carriers was unstable, with half-time of
research, the stability of the biocatalyst also increased after 4.36 and 0.58 h at 508C, respectively.
amination. The partially modified ePGA 1 mM and 5 mM Further optimization of this covalent immobilization pro-
were 1.4- and 6.6-fold more stable than the conventionally cess including chemical amination of PGA led to a stable
Biotechnol. Prog., 2016, Vol. 32, No. 1
immobilized PGA system applicable for the industrial appli-
cation. The half-life of this PGA preparation increased sig-
nificantly depending on the degree of amination: 4.5-fold for
slightly modified pPGA 1 mM, 22.2-fold for partially modi-
fied pPGA 5 mM, and 53.7-fold for highly modified pPGA
10 mM compared with the free enzyme. Similarly, it was
shown that from the aspect of stability of the biocatalyst,
poly(GMA-co-EGDMA) was apparently better support than
Eupergit C.
Acknowledgments
The authors thank the Ministry of Education, Science and
Technological Development of Serbia (Project No. III 46010
and Eureka project E!6750) for the financial support. Also,
the authors are grateful to DSM Anti-Infectives (The Nether-
lands) for providing penicillin G acylase used in this study.
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