Efficient synthesis of cefadroxil in [Bmim][NTf2]-phosphate cosolvent by magnetic immobilized penicillin G acylase

Article abstract of DOI:10.1002/jccs.201900272

For the first time, cefadroxil was synthesized from 7-Amino-3-desacetoxycephalosporanic acid and d-hydroxyphenylglycine methyl ester in [Bmim][NTf₂]-phosphate cosolvent capable of dissolving the substrates using the penicillin G acylase (PGA) immobilized on the micrometer-size magnetic polymer microspheres having high activity of 2,083 U/g. The high synthesis/hydrolysis (S/H) ratio of 1.12 was achieved with 79.0% yield, where only the S/H ratio of 0.19 and yield of 20.0% was obtained using free PGA under the identical optimum reaction conditions. Cefadroxil had been synthesized efficiently in [Bmim][NTf₂]-phosphate cosolvent by the magnetic immobilized PGA, which illuminated that there are two very critical and essential designs, that is, effective support and suitable solvent system by PGA, in enzymatic synthesis of cefadroxil. Obviously, there is great potential for the magnetic immobilized PGA and ionic liquid solvent in application to biocatalysis.

Full text of DOI:10.1002/jccs.201900272

Received: 7 July 2019
Revised: 30 September 2019
Accepted: 30 September 2019
DOI: 10.1002/jccs.201900272
A R T I C L E
Efficient synthesis of cefadroxil in [Bmim][NTf2]-phosphate
cosolvent by magnetic immobilized penicillin G acylase
Zheng Zhaoyu | Hu Chunmiao | Du Chuanhu | Xue Ping | Zhang Weiwei
State Key Laboratory of High-efficiency
Utilization of Coal and Green Chemical
Abstract
Engineering, College of Chemistry &
Chemical Engineering, Ningxia University,
Yinchuan, China
For the first time, cefadroxil was synthesized from 7-Amino-3-desacetoxycephalosporanic
acid and D-hydroxyphenylglycine methyl ester in [Bmim][NTf2]-phosphate
cosolvent capable of dissolving the substrates using the penicillin G acylase (PGA)
immobilized on the micrometer-size magnetic polymer microspheres having high
activity of 2,083 U/g. The high synthesis/hydrolysis (S/H) ratio of 1.12 was
achieved with 79.0% yield, where only the S/H ratio of 0.19 and yield of 20.0%
was obtained using free PGA under the identical optimum reaction conditions.
Cefadroxil had been synthesized efficiently in [Bmim][NTf2]-phosphate cosolvent
by the magnetic immobilized PGA, which illuminated that there are two very criti-
cal and essential designs, that is, effective support and suitable solvent system by
PGA, in enzymatic synthesis of cefadroxil. Obviously, there is great potential for
the magnetic immobilized PGA and ionic liquid solvent in application to
biocatalysis.
Correspondence
Xue Ping, State Key Laboratory of High-
efficiency Utilization of Coal and Green
Chemical Engineering, College of
Chemistry and Chemical Engineering,
Ningxia University, Yinchuan 750021,
China.
Email: ping@nxu.edu.cn
Funding information
National Natural Science Foundation of
China, Grant/Award Numbers: 21263020,
21663020
K E Y W O R D S
[Bmim][NTf2]-phosphate cosolvent, cefadroxil synthesis, micrometer-size magnetic polymer
microspheres, penicillin G acylase, S/H ratio
have been devoted to the study of enzyme-catalyzed synthe-
sis of antibiotics to develop green and efficient synthesis
1 | INTRODUCTION
methods.[3,4] The penicillin G acylase (PGA, EC 3.5.1.11)
has be identified as an important enzyme in the manufacture
of semisynthetic β-lactam antibiotics such as amoxicillin and
cefadroxil because it is capable of hydrolyzing penicillin G
salt to yield a key intermediate 6-aminopenicillanic acid in
the preparation of several β-lactam antibiotics and acylating
semisynthetic intermediate such as 6-aminopenicillanic acid
and 7-amino-3-desacetoxycephalosporanic acid to synthe-
size semisynthetic β-lactam antibiotics under mild reaction
conditions.[5,6] However, free PGA is largely limited in
industry application because of inactivation, low stability,
and hard-to-recycle.[7–9] Accordingly, the immobilization of
PGA on solid supports has been a challenging issue in appli-
cation at all times to enhance its stability and
β-lactam antibiotics, accounting for about 65% on the market
among the current antibiotics, have played significant roles
in the treatment of infectious diseases because of their low
toxicity and good efficacy. As one of the important varieties
of β-lactam antibiotics, cefadroxil is widely used in various
infection treatments due to its high activity, broad
antibacterial spectrum, good oral absorption, and low toxic
side-effects. The industrial production of cefadroxil based
on chemical synthesis encountered tedious and toxic opera-
tion processes, including cumbersome protection and
deprotection as well as use of toxic chlorine reagents so that
generated many nonrecyclable wastes.[1,2] One of effective
ways to solve the above problems is to use biocatalytic syn-
thesis instead of chemical synthesis. So many researchers
© 2019 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J Chin Chem Soc. 2019;1–9.
http://www.jccs.wiley-vch.de
1

2
ZHAOYU ET AL.
reusability.[10–13] Magnetic polymer microspheres comprised
polymers and inorganic magnetic materials, which possessed
sensitive magnetic responsiveness of magnetic nanoparticles
and structural and functional designability of the polymers.
Meanwhile, the biological compatibility of magnetic poly-
mer microsphere surface could be improved by modification
of organic functional groups. In recent years, the magnetic
polymer microspheres have been developing as novel func-
tional materials.[14–16] Wang et al.[17] reported magnetic
poly(styrene-glycidyl methacrylate) porous microspheres
with high magnetic content were prepared by surfactant
reverse micelles and emulsion polymerization of monomers,
in which the well-dispersed Fe3O4 nanoparticles were modi-
fied by polyethylene glycol and oleic acid respectively. Xue
et al.[18] reported that the magnetic hydrophilic polymeric
microspheres containing epoxy groups were prepared by
inverse suspension polymerization of glycidyl methacrylate,
N,N0-methylene bisacrylamide and N-vinyl pyrrolidone in
the existence of formamide dispersed Fe3O4 to obtain the
porous support for immobilizing PGA. These showed that
the surface modification of Fe3O4 nanoparticles using the
special organic compounds is essential to form regular
spherical magnetic polymeric composites to improve the
compatibility between Fe3O4 particles and organic mono-
mers. However, the preparation processes were tedious and
difficult to control the microspheres size and narrow distri-
bution by emulsion polymerization of monomers and sus-
pension polymerization.
consumption of acyl side chain that leads to low production cost.
In fact, there are two reasons for low productivity. On the one
hand, there is significant difference in the optimal pH for complete
dissolution of substrate 7-amino-3-desacetoxycephalosporanic
acid and D-hydroxyphenylglycine methyl ester in aqueous
solution; the former can only be dissolved in extreme alka-
line solution, while the latter can be dissolved in strongly
acidic solution. This leads to partial dissolution of the
substrate 7-amino-3-desacetoxycephalosporanic acid or
D-hydroxyphenylglycine methyl ester at the pH close to neu-
tral of aqueous solution for PGA-catalyzed reaction. On the
other hand, the product cefadroxil and substrate D-
hydroxyphenylglycine methyl ester are easily hydrolyzed by
PGA to produce the by-product D-hydroxyphenylglycine in
aqueous solution (Figure 1). Accordingly, some
researchers were trying to synthesize semisynthetic antibi-
otics in organic solvents to dissolve the substrates and
inhibit hydrolytic side reactions.[20–22] Nevertheless, vola-
tile organic solvents could cause detrimental impact on the
environment and activity of PGA. Therefore, it is neces-
sary to develop alternatives for enzyme-catalyzed synthe-
sis in environmentally friendly reaction media. Ionic
liquids have become a new type of green solvent and reac-
tion medium due to their low volatility, good biological
compatibility, and stability,[23–26] and thus there is great
potential for ionic liquids incorporation in biocatalysis.
But, a specific optimization process, in particular the
immobilization of enzyme and the design of solvent sys-
tem, is needed for each enzymatic process and there is no
one-fits-all solution.
The enzymatic synthesis of cefadroxil antibiotic dis-
played low yield and low synthesis/hydrolysis ratio (the S/H
ratio, the molar ratio of product cefadroxil to by-product D-
phenylglycine),[19] and here, the S/H ratio helps evaluate the
economic viability of the enzymatic process. The key prob-
lem to be solved in enzymatic synthesis of cefadroxil is to
improve the S/H ratio and yield and reduce the redundant
In the present work, the micrometer-size magnetic poly-
mer microspheres were prepared in the presence of magnetic
fluid from dispersed Fe3O4 nanoparticles using natural abun-
dant chitosan having low cost, biodegradability, and bio-
compatibility for immobilizing PGA. For the first time, we
NH
NH
2
2
H N
H
2
S
N
S
PGA-PM@Fe3O4
Synthesis
O
CH3OH
N
O
N
O
O
HO
HO
O
COOH
COOH
7-ADCA
D-HPGM
Cefadroxil
4
P
O
G
A
3
-
Fe
P
y
M
s
@
@
i
H
s
F
y
M
d
e
ly
ro
P
r
3 O
-
o
l
A
NH
s
i
4
d
2
s
G
y
P
H
OH
O
HO
D-HPG
FIGURE 1 Reactions system of enzymatic synthesis for cefadroxil

ZHAOYU ET AL.
3
used
the
cosolvent
composed
of
1-butyl-
into 50 ml of ethanol under ultrasonication for 30 min. The
solution was heated to 60ꢁC in a water bath, and 3 g of
APTES was added into the solution under mechanical stir-
ring. After being stirred for 8 hr, the polymer microspheres
were separated by a magnet and washed several times with
water and ethanol. Then the products were dried in vacuum.
The magnetic spherical support was named as PM@Fe3O4.
3-methylimidazolium bis((trifluoromethyl) sulfonyl imide
([Bmim][NTf2]) and phosphate buffer as the reaction solvent
to inhibit the hydrolysis side reaction. Cefadroxil with the
high S/H ratio and yield could was synthesized in the
cosolvent by the magnetic immobilized PGA. The influenc-
ing factors on the S/H ratio and yield of cefadroxil synthesis
were investigated in the enzymatic reaction system, and the
process conditions were optimized by response surface
method.
2.3 | Preparation of immobilized PGA
The PM@Fe3O4 microspheres of 0.1 g were dispersed into
5 ml of phosphate buffer (0.1 mol/L, pH 7.8), and 0.5 ml of glu-
taraldehyde solution was added in the system. After mechanical
shaking at room temperature for 5 hr, the microspheres were
separated by magnet and washed with buffer for several times.
Then it was immersed in 3 ml of solution containing 1.0 ml of
PGA original solution and 0.5 ml of [Bmim][NTf2] as well as
1.5 ml of phosphate buffer (0.1 mol/L, pH 7.8). After mechani-
cal shaking at 30ꢁC for 12 hr, the microspheres immobilized
with PGA were collected by a magnet and then washed with
buffer three times. The prepared magnetic immobilized PGA
was denoted as PGA-PM@Fe3O4.
2 | EXPERIMENTAL
2.1 | Materials
PGA (862 U/ml) was supplied by Zhejiang Shunfeng Hai-
deer Co., Ltd., China. Glycidyl methacrylate (GMA) and
polyvinyl pyrrolidone (PVP) were from Acros Organics,
USA. D-hydroxyphenylglycine methyl ester (D-HPGM),
7-Amino-3-desacetoxycephalosporanic acid (7-ADCA) and
3-aminopropyltriethoxysilane (APTES) were obtained from
Shanghai Chemical Reagent
I Co., China. 1-butyl-
3-methylimidazolium bis((trifluoromethyl)sulfonyl imide
(IL, [Bmim][NTf2]) was purchased from Lanzhou Institute
of Chemical Physics (Lanzhou, China). Styrene (St),
chitosan (CS), FeCl3ꢀ6H2O, FeCl2ꢀ4H2O, and 2,2-azobis
(2-methyl propionitrile) (AIBN) were provided by Tianjin
Kemer Chemical Reagent Co., Ltd., China. The other
chemicals were of laboratory grade obtained from different
commercial suppliers.
2.4 | Cefadroxil synthesis by PGA-PM@Fe3O4
The enzymatic synthesis of cefadroxil is shown as Figure 1.
In the series–parallel reactions, the reaction of 7-ADCA with
D-HPGM resulted in cefadroxil and methanol, while the
hydrolysis of D-HPGM and cefadroxil could produce by-
product D-phydroxyphenylglycine (D-HPG).
The synthesis of cefadroxil was carried out in [Bmim]
[NTf2]-phosphate cosolvent by the PGA-PM@Fe3O4. In a
typical assay, PGA-PM@Fe3O4 (as-prepared using 0.1 g
PM@Fe3O4) was added to 5 ml of the cosolvent with [Bmim]
[NTf2] concentration of 62.5% (v/v) containing D-HPGM
(90 mmol/L) and 7-ADCA (50 mmol/L), and then the mixture
was incubated at 30ꢁC and 150 r/min. Aliquots were with-
drawn from the reaction mixture at specified time intervals
and diluted with the corresponding mobile phase prior to high
performance liquid chromatography (HPLC) analysis. In addi-
tion, free PGA (using PGA original solution of 1.0 ml) was
used as catalyst for synthesis of cefadroxil under the above-
mentioned identical reaction conditions for comparison.
HPLC analysis. The reactant and product were analyzed
with Shimazu LC-16 HPLC using Agilent TC-C18 chro-
matographic column. The mobile phase was a mixture of
85% (v/v) phosphate sodium buffer (50 mmol/L, pH 5.5)
and 15% (v/v) methanol at a flow rate of 1 ml/min. The
retention time of D-HPG, 7-ADCA, cefadroxil, and D-
HPGM were 4.24, 6.00, 10.87, and 11.50 min, respectively.
All data reported are averages of experiments performed
three times.
2.2 | Preparation of magnetic polymer
microspheres
Forty milligrams of CS was dissolved in 25 ml of an aque-
ous solution of acetic acid (2%). Then, 0.98 g of FeCl3ꢀ6H2O
and 0.48 g of FeCl2ꢀ4H2O was dissolved in the chitosan
solution, and then concentrated aqueous ammonia was
slowly added to adjust the pH to 9 with vigorous stirring.
Under the nitrogen protection, the reaction was carried out at
30ꢁC for 1 hr to form CS-Fe3O4 magnetic fluid. Then,
0.42 g of AIBN was dissolved in a monomer phase con-
sisting of 8 g of styrene and 0.6 g of GMA, and slowly
added to a system containing 1.5 g of PVP, 125 ml of etha-
nol, and 15 ml of water under stirring. The polymerization
was initiated by AIBN under nitrogen at 60ꢁC for 6 hr with
stirring, and the CS-Fe3O4 magnetic fluid was poured into
the reactor and the reaction was continued for 12 hr. After
the completion of polymerization, the magnetic polymer
microspheres were separated by a magnet and washed sev-
eral times with water and ethanol dried in vacuum. Then,
1.5 g the prepared magnetic microspheres were dispersed

4
ZHAOYU ET AL.
2.5 | Characterization of PM@Fe3O4 and
activity assay of PGA-PM@Fe3O4
The morphology of PM@Fe3O4 magnetic microspheres and
CS-Fe3O4 magnetic fluid postclotting was studied by scan-
ning electron microscopy (SEM, JEOL JSM-6360LV) and
transmission electron microscopy (TEM, Hitachi HT7700),
respectively. The magnetization curves were measured using
a vibrating sample magnetometer (VSM, Lake Shore 7304)
with magnetic field intensity in the range of −1.6 × 106 to
1.6 × 106 A/m. X-ray diffraction (XRD, K/MaX-3c) was
used to investigate the crystal composition of PM@Fe3O4.
The fluorescence spectrum of free PGA was determined
using an F-4600 fluorescence spectrophotometer in different
concentrations of [Bmim][NTf2]. Determination of the
epoxy groups on PM@Fe3O4 microspheres was carried out
as follows: 1.0 g of the microspheres was added to 15 ml of
0.65 mol/L sodium thiosulphate solution and the mixture
was maintained in a shaking bed at 30ꢁC for 72 hr, then the
solution was titrated by addition of 0.0921 mol/L
hydrochloric acid until neutralization. The amount of epoxy
groups was calculated from the amount of hydrochloric acid
needed to maintain neutrality of the mixture.[27]
The activity of PGA-PM@Fe3O4 for catalyzing hydroly-
sis of penicillin G natrium to produce 6-aminopenicillanic
acid (6-APA) was determined by the titration method.[28]
The process of measurement was as follows: First, 8 ml of
deionized water and 2 ml of 0.1 mol/L phosphate buffer
solution (0.1 mol/L，pH 7.8) were added to the immobilized
enzyme PGA-PM@Fe3O4 and kept at 37ꢁC in a thermostatic
bath, and then 10 ml of 8% (w/v) aqueous solution of peni-
cillin G natrium kept at 37ꢁC for 5 min was added. The mix-
ture solution was titrated with 0.0990 mol/L NaOH solution
under stirring to maintain the initial pH value. The volume
of NaOH consumed in the first 3 min was recorded, and the
activity of PGA-PM@Fe3O4 was calculated. To investigate
its operational stability, PGA-PM@Fe3O4 was recycled by
magnetic force at the end cycle then used for the next cycle.
The hydrolysis reaction of penicillin G natrium was per-
formed according to above method and repeated in ten
batches. The PGA-PM@Fe3O4 were recovered by magnetic
force and washed twice with phosphate buffer solution
(0.1 mol/L, pH 7.8) between batches.
FIGURE 2 TEM image of CS-Fe3O4 magnetic fluid postclotting
(Figure 2). The result indicated that biopolymer chitosan
with rather good biocompatibility and biodegradability, and
abundant accessibility could become an available dispersant
and surface modifier with potential application for func-
tionalization of inorganic Fe3O4 particles especially in the
preparation of stable magnetic fluids. The PM@Fe3O4
microspheres were obtained by dispersion polymerization
of GMA and St, on which the nanoparticles ferric oxide
coating was formed in situ using the chitosan-dispersed
ferrofluids as modifier and the amino groups introduced
subsequently by the reaction of the hydroxy groups on
the magnetic microspheres with silicon hydroxyl of
3-aminopropyltriethoxysilane. The enzyme molecule could
be immobilized on the magnetic polymer microspheres by
covalent binding between reactive epoxy groups on the sur-
face with the amino group of the enzyme as well as cova-
lent crosslinking the surface-existing amino groups with
glutaraldehyde shown in Figure 3. As a result, we have
achieved successfully the separable and reusable magnetic
biocatalyst PGA-PM@Fe3O4 for cefadroxil synthesis in
ionic liquid-phosphate cosolvent.
Figure 4a shows that the PM@Fe3O4 has a perfect spher-
ical shape and the average size of 2 μm with smooth outer
surface. The FTIR spectrum of PM@Fe3O4 magnetic micro-
spheres is shown in Figure 4b. The absorption peak located
at 636 cm−1 is the characteristic absorption of Fe3O4, which
showed the presence of Fe3O4 particles in polymer micro-
spheres. The absorption peak located at 904 cm−1 is the
characteristic absorption of the epoxy groups.[29,30] The
results of chemical analysis indicated that PM@Fe3O4 had
epoxy groups of 0.11 mM/g. The peak at 3,438 cm−1 was
attributed to the absorption of amine groups on the
PM@Fe3O4 magnetic microspheres. The peaks at 1,032 and
701 cm−1 belonged to the stretching vibration of saturated
Si-O bonds and Si-C bonds, respectively. The analytical
3 | RESULTS AND DISCUSSION
3.1 | Characterization of PM@Fe3O4
magnetic microspheres
The designed and prepared PM@Fe3O4 magnetic support
for immobilizing PGA possesses the multiple functional
actions. It was observed that the generated Fe3O4 in situ was
dispersed highly into the chitosan film in the ferrofluids

ZHAOYU ET AL.
5
St
NH
2
OH
APTES
Fe O -CS magnetic fluid
2
3
4
H
C
O
NH
GMA
AIBN
CH
OH
2
H
C
H
C
CH
2
CH
2
O
O
C
H
N
EnZ
CHO
OHC(CH ) CHO
2 3
PGA
C
H
N
EnZ
EnZ
CHO
H
C
H
CH
C
N
C
2
H H
2
O
OH
FIGURE 3 Preparation diagram of PM@Fe3O4 and PGA-PM@Fe3O4
(a)
(a)
311
220
30
440
511
422
400
10
20
40
50
60
70
(b)
2ꢀ/degree
(b)
15
904
1032
10
5
636
3438
0
-5
-10
701
500
4000 3500 3000 2500 2000 1500 1000
-15
Wavenumbers (cm-1)
-15000
-10000
-5000
0
5000
10000
15000
H(Oe)
FIGURE 4 SEM image (a) and FTIR spectrum (b) of
PM@Fe3O4
FIGURE 5 XRD pattern (a) and hysteresis loops (b) of
PM@Fe3O4
results of FTIR spectrum confirmed that the PM@Fe3O4
magnetic microspheres contained the reactive epoxy groups
and amine groups bonding the enzyme molecules.
(511), and (440) planes of Fe3O4, respectively.[31] It could
be seen that the magnetic microspheres could been prepared
conveniently and efficiently using the ferrofluids dispersed
in a natural chitosan solution from the magnetic source of
Fe3+and Fe2+ ions instead of solid Fe3O4. The broadening
peak at 2θ in 15ꢁ–20ꢁ in the XRD pattern belongs to the
amorphous polymeric components in PM@Fe3O4 magnetic
The XRD pattern of PM@Fe3O4 magnetic microspheres
is shown in Figure 5a. The results showed that Fe3O4 parti-
cles in PM@Fe3O4 magnetic microspheres were in an
inverse cubic spinel structure, which exhibited characteristic
peaks at 2θ of 30.1ꢁ, 35.6ꢁ, 43.5ꢁ, 53.8ꢁ, 57.4ꢁ, and 62.9ꢁ
corresponding to the indices of (220), (311), (400), (422),

6
ZHAOYU ET AL.
TABLE 1 The S/H ratio and Yield of synthesizing cefadroxil in
different solvents
2000
1500
1000
500
0
Solvent
Yield (%)
6.6
S/H ratio
0.09
Dichloromethane
Phosphate buffer (0.1 mol/L, pH 7.8)
[Bmim][NTf2]
5.6
0.08
15.6
0.30
Note: Reaction conditions: 50 mmol/L 7-ADCA, 90 mmol/L D-HPGM,
30ꢁC, 18 hr.
7-ADCA and D-HPGM in phosphate buffer and serious
hydrolysis of D-HPGM. The cefadroxil yield of 6.6% and
the S/H ratio of 0.09 were achieved in dichloromethane sol-
vent. As shown in Table 1, while using ionic liquid [Bmim]
[NTf2] capable of dissolving the substrates as solvent in the
enzyme-catalyzed reaction, the PGA-PM@Fe3O4 showed
best catalytic efficiency compared with those in the above
solvents, the S/H ratio and yield reached 15.6% and 0.30,
respectively. Nevertheless, the high solvent viscosity of the
ionic liquid created serious resistance to the diffusion of
reactant molecules leading to a reduction in catalytic effi-
ciency. Hence, we have explored [Bmim][NTf2]-phosphate
cosolvent system used in synthesis of cefadroxil to improve
catalytic efficiency of the PGA-PM@Fe3O4 to a greater
degree.
1
2
3
4
5
6
7
8
9
10
Recycle numbers
FIGURE 6 Activity and operational stability of PGA-
PM@Fe3O4
microspheres. A typical magnetization curve for PM@Fe3O4
is shown in Figure 5b in which there is no hysteresis in the
magnetization with both remanence and coercivity being
zero as well as 13.0 emu/g of saturation magnetization.
Therefore, the microspheres display superparamagnetic
behavior, and their separation in liquid media can be con-
trolled by magnetic forces.
The test results of activity and operational stability of
PGA-PM@Fe3O4 for catalyzing hydrolysis of penicillin G
natrium to produce 6-APA were listed in Figure 6. It was
obtained that the PGA immobilized on PM@Fe3O4 mag-
netic microspheres had high catalytic activity of 2,083 U/g,
which showed that the microspheres could provide a good
microenvironment for the immobilization of enzyme and
express of its catalytic activity. The PGA-PM@Fe3O4
microspheres were used for consecutive cycles to catalyze
hydrolysis of penicillin G natrium to 6-APA, and its activity
was 1,918 U/g retaining 92.1% of the initial activity after
10 cycles, indicating for the PGA-PM@Fe3O4 had consider-
able operational stability. The results could illuminate that
the PM@Fe3O4 magnetic microspheres were excellent sup-
port for immobilizing PGA duo to covalent immobilization
by means of the coupling between the amino groups of the
enzyme and the epoxy groups existing on the microspheres
as well as covalent crosslinking the surface-existing amino
groups with glutaraldehyde.
3.3 | Cefadroxil synthesis in [Bmim][NTf2]-
phosphate cosolvent
The effect of [Bmim][NTf2] volume concentration in the
cosolvent on the S/H ratio and yield of cefadroxil by PGA-
PM@Fe3O4 was investigated under the experimental condi-
tions of 50 mmol/L 7-ADCA and 90 mmol/L D-HPGM at
30ꢁC for 18 hr synthesis. It was observed that there were
significant increases of the S/H ratio and yield for cefadroxil
synthesis in [Bmim][NTf2]-phosphate cosolvent by PGA-
PM@Fe3O4 from Figure 7, which indicated that [Bmim]
[NTf2] existing in the solution was advantageous in improv-
ing the synthesis activity of PGA-PM@Fe3O4 in full play.
The S/H ratio and yield increased with the increase of con-
centration of the [Bmim][NTf2] until they reached the maxi-
mum value of 79.0% and 1.12 at [Bmim][NTf2]
concentration of 62.5% (v/v). As the concentration of
[Bmim][NTf2] was on the increase the S/H ratio and yield
declined gradually because of increase in solvent viscosity
of the PGA-PM@Fe3O4 catalyzed reaction system.
3.2 | Cefadroxil synthesis by PGA-PM@Fe3O4
in different solvents
The analytical results by fluorescence spectroscopy found
that the maximum of fluorescence intensity of excitation
spectrum or the emission spectrum of free PGA occurred at
the [Bmim][NTf2] concentration of 62.5% (v/v) as shown
Figure 8, and the variation of the fluorescence intensity was
consistent with that of the synthesis activity of PGA-
The yield and the S/H ratio of synthesizing cefadroxil by
PGA-PM@Fe3O4 in different solvents were listed in
Table 1. In phosphate buffer (0.1 mol/L, pH 7.8), the reac-
tion product cefadroxil could not be detected nearly, which
may be attributed to the poor solubility of substrate

ZHAOYU ET AL.
7
(a) 1.4
1.2
(a)
100
80
60
40
20
0
1
7
1.0
0.8
0.6
0.4
0.2
390 420 450 480 510 540 570
Wavelength(nm)
0.0
0
20
40
60
80
100
[Bmim][NTf2] (v/v)
(b) 350
300
250
200
150
100
50
90
80
70
60
50
40
30
20
10
0
(b)
1
7
0
410 415 420 425 430 435 440 445 450
Wavelength(nm)
0
20
40
60
80
100
[Bmim][NTf2](v/v)
FIGURE 8 Excitation spectra (a) and emission spectra (b) of free
PGA in different concentrations of [Bmim][TNf2]-phosphate buffer
1–7 stands for C[Bmim][TNf2](v/v): 62.5, 75, 50, 37.5, 25, 100,
and 0%
FIGURE 7 Effect of [Bmim][NTf2] concentration on S/H ratio
(a) and yield (b) of cefadroxil synthesis
PM@Fe3O4 in the cosolvent with different concentrations of
[Bmim][NTf2]. The intrinsic fluorescence spectrum is
mainly attributed to aromatic amino acids (Trp, Tyr, and
Phe) on side chains of the tertiary structure of enzyme pro-
tein.[32,33] Therefore, the variation of fluorescence intensity
for PGA could reflect the degree of changes in its tertiary
structure in different concentration of [Bmim][NTf2] in the
cosolvent. The fluorescence intensity of excitation or emis-
sion for PGA in [Bmim][TNf2]-phosphate buffer cosolvent
was much higher than that in phosphate buffer. That could
mean the spatial conformation of the enzyme in [Bmim]
[TNf2]-phosphate buffer cosolvent became in a relatively
stretched state suitable for activity expression of the enzyme.
Accordingly, the activity of PGA was enhanced in the
[Bmim][TNf2]-phosphate cosolvent. While the concentration
was 62.5%, the fluorescence intensity reached the maximum,
the catalytic activity of the magnetic immobilized PGA for
synthesizing cefadroxil was the highest as well. When the
concentration of ionic liquids was 75% or 100%, the fluores-
cence intensity for PGA decreased. It may be attributed to
the destruction of the active center due to the overstretching
of the whole structure, and the activity of PGA decreased
accordingly.
3.4 | Effect of temperature and time on
cefadroxil synthesis
Controlling 50 mmol/L of 7-ADCA, 90 mmol/L of D-
HPGM, and reaction temperature range from 20 to 40ꢁC,
cefadroxil was synthesized by PGA-PM@Fe3O4 in the
cosolvent with [Bmim][NTf2] concentration of 62.5%. As
shown in Table 2, the S/H ratio and yield were significantly
affected by reaction temperature and time. The S/H ratio of
1.01 and yield of 76.7% were obtained from the reaction at
40ꢁC for 12 hr, while the S/H ratio and yield were 1.07 and
73.7% at 20ꢁC for 28 hr, respectively. Overall, the S/H ratio
and yield reached the maximum of 79.0% and 1.12 at 30ꢁC
after 18 hr reaction. Therefore, the appropriate reaction tem-
perature and time appeared to be 30ꢁC and 18 hr for synthe-
sizing cefadroxil by PGA-PM@Fe3O4.

8
ZHAOYU ET AL.
TABLE 2 The S/H ratio and yield of
cefadroxil synthesis at different
temperatures
20ꢁC
30ꢁC
40ꢁC
Time (hr)
Yield (%)
29.1
S/H ratio
0.32
Yield (%)
35.1
S/H ratio
0.46
Yield (%)
57.5
S/H ratio
0.71
4
8
36.7
0.45
48.9
0.65
68.0
0.82
12
16
18
24
28
50.2
0.58
59.6
0.75
76.7
1.01
64.0
0.78
70.2
1.01
69.8
0.98
70.2
0.94
79.0
1.12
64.5
0.84
73.6
1.05
75.5
1.03
55.6
0.7
73.7
1.07
67.8
0.86
50.7
0.66
Note: Reaction conditions: 50 mmol/L 7-ADCA, 90 mmol/L D-HPGM, cosolvent of 62.5% [Bmim][NTf2].
TABLE 3 Effect of molar ratio of D-HPGM to 7-ADCA on
TABLE 4 Comparative study between synthetic performance of
cefadroxil synthesis
free PGA and PGA-PM@Fe3O4
Mole ratio (D-HPGM/7-ADCA)
Yield (%)
42.5
S/H ratio
0.57
Catalyst
Yield (%)
20.0
S/H ratio
0.19
1:1
Free PGA
1.4:1
1.8:1
2.2:1
2.6:1
74.8
0.95
PGA-PM@Fe3O4
79.0
1.12
79.0
1.12
Note: Reaction conditions: 50 mmol/L 7-ADCA, 90 mmol/L D-HPGM,
cosolvent of 62.5% [Bmim][NTf2], 30ꢁC, 18 hr.
76.7
0.74
74.4
0.66
Note: Reaction conditions: 50 mmol/L 7-ADCA, cosolvent of 62.5% [Bmim]
[NTf2], 30ꢁC, 18 hr.
(79.0% vs 20.0%) and the S/H ratio (1.12 vs 0.19) than free
PGA as catalyst, which indicated that the PGA immobilized
on the PM@Fe3O4 magnetic polymer microspheres pos-
sesses excellent catalytic performance for cefadroxil synthe-
sis in [Bmim][NTf2]-phosphate cosolvent. Furthermore,
PGA-PM@Fe3O4 can be recycled easily under a magnetic
field.
3.5 | Effect of molar ratio D-HPGM to
7-ADCA on cefadroxil synthesis
The influence of molar ratios of D-HPGM to 7-ADCA on
the S/H ratio and yield of cefadroxil by the PGA-
PM@Fe3O4 was investigated in the cosolvent with [Bmim]
[NTf2] concentration of 62.5% and the results were listed in
Table 3. When the molar ratio of D-HPGM to 7-ADCA
increased from 1:1 to 1.8:1, the S/H ratio increased from
0.57 to 1.12, and the yield rose from 42.5 to 79.0%. But the
both reduced in increase further of the molar ratio of D-
HPGM to 7-ADCA，and there was a maximum of the S/H
ratio (1.12) or yield (79.0%) at the molar ratio of 1.8:1. Usu-
ally, the semisynthetic reaction had been carried out with a
high substrates molar ratio of 2:1–4:1 in order to obtain the
relatively high yield of semisynthetic β-lactam antibiotics
products.[34] However, the much excessive acyl lateral was
used leading to the poor utilization of the raw materials. It
was found that at relatively low substrates molar ratio of
1.8:1 cefadroxil could been synthesized with the high S/H
ratio and yield in [Bmim][TNf2]-phosphate cosolvent by the
as-preparation PGA-PM@Fe3O4.
4 | CONCLUSIONS
The magnetic polymer microspheres with the average size of
2 μm and saturation magnetization of 13.0 emu/g were pre-
pared in the presence of the chitosan-dispersed ferrofluids in
situ and employed as support for immobilizing PGA. The
magnetic spherical biocatalyst was obtained by covalent
coupling between the amino groups of PGA and epoxy
groups existing on the microspheres as well as covalent
crosslinking the surface-existing amino groups with glutar-
aldehyde. The magnetic immobilized PGA was used for
efficient synthesis of cefadroxil in [Bmim][NTf2]-phosphate
cosolvent capable of dissolving substrate 7-ADCA along
with D-HPGM, and the S/H ratio of 1.12 and yield of
79.0% were achieved. The both were far above the results
in the case of using free PGA under the identical optimum
reaction conditions. Obviously, the magnetic immobilized
PGA is an efficient, easily separated, and recycled biocata-
lyst for biosynthetic reactions in promising industrial
application.
A comparative study between the catalytic performance
of free PGA and PGA-PM@Fe3O4 was conducted, and the
result was shown in Table 4. The PGA-PM@Fe3O4-catalytic
cefadroxil synthesis exhibited considerably higher yield

ZHAOYU ET AL.
9
[19] M. A. Wegman, M. H. A. Janssen, F. van Rantwijk,
R. A. Sheldon, Adv. Synth. Catal. 2001, 343, 559.
[20] A. L. Deaguero, A. S. Bommarius, Biotechnol. Bioeng. 2014,
111, 1054.
ACKNOWLEDGMENTS
This work was financially supported by the National Natural
Science Foundation of China (No. 21663020, 21263020).
[21] R. Fernandezlafuente, C. M. Rosell, J. M. Guisán, Enzyme
Microb. Technol. 1991, 13, 898.
[22] D. Roche, K. Prasad, O. Repic, Tetrahedron. Lett. 1999, 40,
3665.
[23] M. Goldfeder, A. Fishman, Appl. Microbiol. Biotechnol. 2014,
98, 545.
REFERENCES
[1] C. G. P. H. Schroen, V. A. Nierstrasz, P. J. Kroon, R. Bosma,
A. E. M. Janssen, H. H. Beeftink, J. Tramper, Enzyme Microb.
Technol. 1999, 24, 498.
[24] T. Itoh, Chem. Rev. 2017, 117, 10567.
[2] S. C. Pereira, R. Bussamara, G. Marin, R. L. C. Giordano,
J. Dupont, R. de Campos Giordano, Green Chem. 2012, 14, 3146.
[3] Q. Wu, C. X. Chen, L. L. Du, X. F. Lin, Appl. Biochem. Bio-
technol. 2010, 160, 2026.
[4] S. W. Deng, E. Z. Su, X. Q. Ma, S. L. Yang, D. Z. Wei,
J. Biotechnol. 2015, 199, 62.
[25] M. A. Gebbie, A. M. Smith, H. A. Dobbs, A. A. Lee, G. G. Warr,
X. Banquy, M. Valtiner, M. W. Rutland, J. N. Israelachvili,
S. Perkin, R. Atkin, Chem. Commun. 2017, 53, 1214.
[26] Y. H. Bi, Z. Q. Duan, X. Q. Li, Z. Y. Wang, X. R. Zhao, J. Agr.
Food. Chem. 2015, 63, 1558.
[27] L. Sundberg, J. Porath, J. Chromatogr. 1974, 90, 87.
[28] P. Xue, G. Z. Lu, Y. L. Guo, Y. S. Wang, Y. Guo, J. Mol. Catal.
B: Enzym. 2004, 30, 75.
[29] Z. D. Knezevic-Jugovic, M. G. Zuza, S. M. Jakovetic,
A. B. Stefanovic, E. S. Dzunuzovic, K. B. Jeremic,
S. M. Jovanovic, Biotechnol. Progr. 2016, 32, 43.
[30] G. Bayramoglu, Y. Tunali, M. Y. Arica, Catal. Commun. 2007, 8,
1094.
[31] Z. F. Ali, L. Tian, P. P. Zhao, B. L. Zhang, A. Nisar, X. J. Li,
H. P. Zhang, Q. Y. Zhang, RSC Adv. 2015, 5, 92449.
[32] B. Wang, Y. Y. Zhang, C. Venkitasamy, B. G. Wu, Z. L. Pan,
H. L. Ma, Food. Chem. 2017, 234, 20.
[5] P. Xue, Y. H. Gu, W. G. Su, H. H. Shuai, J. L. Wang, Appl. Surf.
Sci. 2016, 362, 427.
[6] S. W. Deng, X. Q. Ma, M. Sun, D. Z. Wei, E. Z. Su, Catal.
Commun. 2016, 79, 31.
[7] X. Pan, L. Wang, J. Ye, S. Qin, B. He, Appl. Microbiol. Biot.
2018, 102, 1749.
[8] S. A. Ansari, Q. Husain, Biotechnol. Adv. 2012, 30, 512.
[9] S. Jun, J. Lee, B. C. Kim, J. E. Lee, J. Joo, H. Park, J. H. Lee,
S. Lee, D. Lee, S. Kim, Y. Koo, C. H. Shin, S. W. Kim,
T. Hyeon, J. Kim, Chem. Mater. 2012, 24, 924.
[10] B. L. Zhang, J. Q. Wang, J. J. Chen, H. P. Zhang, D. Z. Yin,
Q. Y. Zhang, Biochem. Eng. J. 2017, 127, 43.
[33] Y. C. Fan, S. L. Zhang, Q. Wang, J. H. Li, H. T. Fan, D. K. Shan,
Appl. Spectrosc. 2013, 67, 648.
[11] L. L. Fan, B. L. Zhang, H. P. Zhang, X. K. Jia, X. C.,.
Q. Y. Zhang, RSC Adv. 2016, 6, 65911.
[34] Z. X. Huang, S. L. Cao, P. Xu, H. Wu, M. H. Zong, W. Y. Lou,
Chem. Eng. J. 2018, 346, 361.
[12] P. Xue, C. M. Hu, X. H. Yan, G. L. Fang, H. F. Shen, J. Chin.
Chem. Soc. 2018, 66, 427.
[13] C. Y. Chen, Y. H. Lu, J. T. Lin, C. C. Hu, C. B. Fuh, H. Y. Tsai,
J. Chin. Chem. Soc. 2018, 65, 1075.
[14] B. Xu, C. Zheng, H. Zheng, Y. Wang, C. Zhao, C. Zhao,
S. Zhang, RSC Adv. 2017, 7, 47029.
How to cite this article: Zhaoyu Z, Chunmiao H,
Chuanhu D, Ping X, Weiwei Z. Efficient synthesis of
cefadroxil in [Bmim][NTf2]-phosphate cosolvent by
magnetic immobilized penicillin G acylase. J Chin
Chem Soc. 2019;1–9. https://doi.org/10.1002/jccs.
201900272
[15] X. Y. Liu, S. W. Zheng, R. Y. Hong, Y. Q. Wang, W. G. Feng,
Colloids Surf. A. Physicochem. Eng. Asp. 2014, 443, 425.
[16] Y. H. Gu, P. Xue, J. Chin. Chem. Soc. 2018, 65, 696.
[17] X. Y. Wang, Z. M. Zhou, G. H. Jing, Bioresour. Technol. 2013,
130, 750.
[18] P. Xue, W. G. Su, Y. H. Gu, H. F. Liu, J. L. Wang, J. Magn.
Magn. Mater. 2015, 378, 306.