Yixiao Fan1,2

Efficient enzymatic synthesis of cephalexin in Yingbo Li1

∗

1∗
suspension aqueous solution system Qingfen Liu

1 CASKey Laboratory of Green Process and Engineering, Institute of

Process Engineering, Chinese Academy of Sciences, Beijing, People’s
Republic of China
2 School
of Chemical Engineering, University of Chinese Academy of
Sciences, Beijing, People’s Republic of China

Abstract
An efficient method for the enzymatic synthesis of cephalexin
(CEX) from 7-amino-3-deacetoxycephalosporanic acid
(7-ADCA) and D-phenylglycine methyl ester (PGME) using
immobilized penicillin G acylase (IPGA) as catalyst in a
suspension aqueous solution system was developed, where
the reactant 7-ADCA and product CEX are mainly present as
solid particles. The effects of key factors on the enzymatic
synthesis were investigated. Results showed that continuous
maximum 7-ADCA conversion ratio of 99.3% and productivity
of 200 mmol/L/H were achieved, both of which are much
superior to the homogeneous aqueous solution system.
Besides, IPGA still retained 95.4% of its initial activity after 10
cycles of enzymatic synthesis, indicating the excellent stability
of this approach. The developed approach shows great
potential for the industrial production of CEX via an
enzyme-based route.  C 2020 International Union of Biochemistry and

feeding of PGME was more efficient for the synthesis of CEX Molecular Biology, Inc. Volume 00, Number 0, Pages 1–12, 2020
than the batch mode. Under the optimized conditions, the

Keywords: cephalexin, enzymatic synthesis, penicillin G acylase,

suspension aqueous solution system, β-lactam antibiotics

1. Introduction reactive groups, hazardous reagents, and harsh reaction condi-

Since bacteriologist Alexander Fleming discovered penicillin
in 1928, β-lactam antibiotics have made a tremendous contri-
bution in fighting against numerous fatal bacteria [1]. Today,
β-lactam antibiotics, especially semisynthetic penicillins and
cephalosporins, still rank at the leading position in total antibi-
otic sales for treating a variety of bacterial-infection diseases
[2]. The industrial production of semisynthetic β-lactam antibi-
otics by a chemical method involves protection/deprotection of
Abbreviations: 7-ADCA, 7-amino-3-desacetoxy cephalosporanic acid;
6-APA, 6-aminopenicillanic acid; CEX, cephalexin; CR, conversion ratio;
HPLC, high-performance liquid chromatography; HR, hydrolysis ratio; IPGA,
immobilized penicillin G acylase; P, productivity; PG, D-phenylglycine; PGA,
penicillin G acylase; PGME, D-phenylglycine methyl ester..
∗ Address for correspondence: Qingfen Liu, PhD and Yingbo Li, PhD ,
Institute of Process Engineering, Chinese Academy of Sciences, Beijing
100049, People’s Republic of China. Tel./Fax: +86-010-82545858; e-mail:
qfliu@ipe.ac.cn; ybli@ipe.ac.cn
Additional supporting information may be found online in the Supporting
Information section at the end of the article.
Received 21 September 2019; accepted 21 February 2020
DOI: 10.1002/bab.1903
Published online in Wiley Online Library
(wileyonlinelibrary.com)
tions, consequently generating a large amount of nonrecyclable
waste and bringing enormous stress to the environment [3].
Alternatively, their enzymatic synthesis has received great
attention as a green technology since the first report on the
reversibility of penicillin acylase–catalyzed hydrolysis of ben-
zylpenicillin [4].
Enzymatic synthesis of β-lactam antibiotics such as
cephalexin (CEX), amoxicillin, and ampicillin employing peni-
cillin G acylase (PGA; E.C. 3.5.1.11) as a biocatalyst can be
conducted under thermodynamic or kinetic control. Unfortu-
nately, the former is generally unfavorable despite great effort
made in the past years. In contrast, the latter is preferred
because reactants’ conversion is not restrained by reaction
equilibrium and higher yield could be achieved [5]. The kinet-
ically controlled synthesis of β-lactam antibiotics is a complex
process, where PGA can act either as a transferase or as a hy-
drolase, catalyzing two undesired side reactions: the hydrolysis
of the acyl side-chain precursor (an ester or amide, a parallel
reaction) and the hydrolysis of the antibiotic itself (a consec-
utive reaction) [6]. For example, when CEX is enzymatically
synthesized from d-phenylglycine methyl ester (PGME) and
7-amino 3-desacetoxy cephalosporanic acid (7-ADCA), PGA
does not only transfer the acyl group of PGME to 7-ADCA to
generate CEX, but also hydrolyze PGME into d-phenylglycine

1

(PG) and CEX into 7-ADCA and PG (Fig. 1A). Therefore, a
high PGME/7-ADCA molar ratio is often used to obtain a high
conversion of 7-ADCA to CEX. However, the superfluous PGME
consumption would not only increase pollutant emissions but
increase difficulty in the downstream processing [7]. In another
aspect, the productivity of CEX is low due to the low solubility
of 7-ADCA in an aqueous medium (the solubility of 7-ADCA is
18–78 mmol/L at pH 6.0–7.0) [8].
To improve the efficiency of penicillin G acylase-catalyzed
synthesis of CEX based on the kinetically controlled method,
great efforts have been made by researchers in the past
decades: optimization of reaction pH and temperature [9–12],
use of cosolvents to reduce the water activity [13–16], in situ
product removal by an aqueous two-phase system [17–20] or
complexation [21,22], modification of the catalytic properties
of the enzyme [23–25], utilization of highly condensed systems
without the aqueous phase [26], as well as control of reactant
concentrations [27–32]. The latter has been extensively studied,
and it is demonstrated that high concentrations of reactants
are beneficial to the conversion ratio and productivity. In
many cases, however, high PGME/7-ADCA molar ratio and low
conversion ratio are still the inherent problem in industrial
production of CEX through enzymatic synthesis.
Enzymatic synthesis in suspension aqueous solution sys-
tems (or called heterogeneous aqueous systems) is an attractive
approach, which has been extensively investigated for the syn-
thesis of numerous products including peptide [33], optically
active d-amino acids [34], and Z-aspartame [35]. In these
systems, most of reactants are suspended in a low-volume,
saturated aqueous phase. As the reaction proceeds, the solid
reactants constantly are dissolved into the aqueous phase
and are converted into soluble or sparingly soluble product(s)
[36–38]. Such systems are particularly suitable for enzymatic
reactions with reactants having low solubility, offering sur-
prisingly high conversion ratio and productivity. Pioneering
work on the enzymatic synthesis of semisynthetic β-Lactam
antibiotics, for example, ampicillin, has been conducted by
Youshko et al. [39], in which ampicillin synthesis proceeded
in 93% conversion on 6-aminopenicillanic acid (6-APA) and in
60% conversion on PGME. However, in that work, the products
in a solid phase consist of both ampicillin and PG, leading to
the disadvantage in the subsequent separation. Moreover, the
factors influencing the enzymatic synthesis have not yet been
investigated.
In this work, the feasibility of enzymatic synthesis of CEX
in a suspension aqueous solution system was explored using 7-
ADCA and PGME as reactants and IPGA as biocatalyst. First, to
reduce the hydrolysis of PGME, its adding manner was studied.
To develop a competitive strategy for enzymatic high-efficiency
synthesis of CEX, factors such as reaction pH, temperature,
enzyme loading, PGME feeding time, amount of water, and
PGME/7-ADCA molar ratio on the enzymatic synthesis were
investigated systematically. Under the optimized conditions,
the performance for enzymatic synthesis of CEX in suspension
aqueous solution and in homogenously aqueous solution was
2 Efficient Enzymatic Synthesis of Cephalexin
Biotechnology and
Applied Biochemistry
compared and discussed. Finally, the recycling stability of the
employed enzyme was probed.
Highlights
r An aqueous solution system was developed for enzymatic
synthesis of cephalexin.
r The influences of key factors on this reaction were ana-
lyzed.
r A conversion ratio of 99.3% for 7-amino 3-desacetoxy
cephalosporanic acid (7-ADCA) was obtained at a very low
d-phenylglycine methyl ester/7-ADCA molar ratio.
r High productivity was achieved at very high initial 7-ADCA
concentrations.
2. Materials and Methods
2.1. Materials
Polyacrylamide gel surface-bound penicillin acylase from
Escherichia coli with enzymatic activity of 152.3 U/g (wet
weight) at pH 7.0 and 20 ◦ C was obtained from Hunan Flag
Biotech Co. Ltd (Hunan, China). The immobilized biocatalyst
spherical particles were yellow color and had a diameter of
approximately 0.25 mm. PGME (99.0%) was purchased from
Hebei Province Chemical Industry Research Institute (Hebei,
China). 7-ADCA (99.9%) and CEX (99.9%) were purchased
from North China Pharmaceutical Co. Ltd (Hebei, China). PG
was purchased from National Institutes for Food and Drug
Control (Beijing, China). Methanol of the chromatographic
grade for analytical use was purchased from Merck(Darmstadt,
Germany). All other chemicals and buffer components were of
analytical grade and were purchased from Sinopharm Chemical
Reagent Co. Ltd. (Beijing, China).
2.2. Enzymatic synthesis of CEX with batch mode or
continuous feeding of PGME
The enzymatic synthesis of CEX was performed in a 500-mL
scale stirred reactor equipped with a stirring paddle, stirring
controller, pH meter, and thermostatic bath as shown in Fig. S1
in the Supporting Formation.
The enzymatic synthesis of CEX with a batch mode of PGME
was operated as follows: First, appropriate amounts of 7-ADCA,
PGME, and deionized water were added into the reactor. The
stirrer was started, and the temperature was controlled at a
certain value. Then, its pH was adjusted to a required value with
3 mol/L ammonia solution. The 7-ADCA suspension aqueous or
homogeneous aqueous solution system was obtained depending
on the adding of 7-ADCA. After incubation for 0.5 H, the reaction
was initiated by adding appropriate amounts of IPGA into the
reaction system. During the synthesis, the pH value of the
reaction system was monitored by a pH meter. When the pH
value was different from the desired pH value, the stock solution
of 3 mol/L ammonia or 6 mol/L hydrochloric acid was added into
the reaction solution by a flow volume control unit. The added
volume of acid or alkali solution was recorded by this flow
 Reaction scheme for enzymatic synthesis of CEX
FIG. 1 catalyzed by PGA in homogeneous aqueous
solution system (A) and suspension aqueous
solution system (B).
volume control unit. Therefore, the total volume of the reaction
solution can be obtained by the original volume of reaction
solution plus the added volume of acid or alkali solution.
Enzymatic synthesis of CEX with continuous feeding of
PGME was carried out as the reaction with the batch mode of
PGME except for adding of PGME and IPGA. Briefly, appropriate
amounts of 7-ADCA and deionized water were first added into
the reactor to generate a suspension solution. The stirrer was
started, and the temperature was controlled at a certain value.
Then, its pH was adjusted to the required value with 3 mol/L
ammonia solution, and appropriate amounts of IPGA were
added into the reactor. After the 7-ADCA suspension aqueous
solution system was incubated for 0.5 H, the reaction was
initiated by feeding the PGME solution with a concentration of
1.65 mol/L into the suspension solution at a constant speed.
Biotechnology and Applied Biochemistry
During the synthesis, the pH of the solution was kept constant
using 3 mol/L ammonia solution and 6 mol/L hydrochloric acid
solution. The changes in the volume of reaction mixture due to
the addition of ammonia solution and hydrochloric acid solution
were also considered by the procedure described above.
Once the adding of PGME was established, a total of six
process parameters that would affect the performances of
the enzymatic synthesis were systematically studied. These
parameters and their tested ranges were as follows: (1) pH
(6.5–7.3); (2) temperature (10–25 ◦ C); (3) enzyme loading
(15.23–38.08 IU/mmol); (4) feeding time of PGME (0.67–1.67
H); (5) initial concentration of 7-ADCA (532–746 mmol/L); (6)
PGME/7-ADCA molar ratio (1.04–1.16 mmol/mmol).
To characterize the change of each composition in reaction
mixtures during the course of enzymatic synthesis, samples
were withdrawn at intervals. Owing to the presence of solid
reactant (7-ADCA) or CEX or PG in the suspension reaction
system, two samples were taken and analyzed according to a
number of studies [30, 32, 39]: One was filtered by a 0.22-µm
membrane to remove the solid particles, and the composition
3

of the resultant filtrate was analyzed; the other aliquot of the
heterogeneous system was taken for characterization of the
whole reaction mixture. The synthesis was evaluated in terms
of conversion ratio (CR), productivity (P), and hydrolysis ratio
(HR).
CR was determined as the ratio of the maximum amount
of CEX (mmol) to initially added amount of 7-ADCA (mmol):
ct,CEX × Vt
CR = × 100% (1)
n0,7-ADCA
where P is defined as the mmol of CEX produced per unit time
and unit reaction volume at the maximum CR.
ct,CEX
P= (2)
t
where HR is defined as the ratio of the amount of hydrolyzed
PG (mmol) to the total amount of added PGME (mmol) at the
maximum CR:
ct,PG × Vt
HR = × 100% (3)
n0,PGME
where n0,7-ADCA is the initially added amount of 7-ADCA (mmol).
n0,PGME is the total amount of added PGME (mmol), Ct,CEX is
the total concentration of CEX at time t (H), Ct,PG is the total
concentration of PG at time t (H), t is the time when the
maximum total concentration of CEX was reached, and Vt is
the total volume of reaction mixtures at time t ( in hours).
2.3. Recycling stability of IPGA
Repeated batch reactions for recycling stability of IPGA in
the suspension aqueous solution were carried out in a 500-
mL reaction apparatus as described in Section 2.2. After the
reaction under the optimal conditions was completed, the
reaction mixture was separated by the 100-mesh sieve and
the IPGA remaining behind was washed three times with fresh
deionized water. The isolated IPGA was added to the fresh
reaction mixture to catalyze the next reaction.
2.4. High-performance liquid chromatography
analysis
Concentrations of CEX, PGME, 7-ADCA, and PG in the
reaction system were determined by high-performance
liquid chromatography (HPLC; Agilent 1260, Califor-
nia, USA) with a reversed-phase column C18 (150 mm
× 4.6 mm, 5 µm; Agilent, USA). The column tempera-
ture was maintained at 30 ◦ C. The mobile phase con-
sisted of sodium dihydrogen phosphate buffer solution
(20 mmol/L, pH 5.0) and methanol. The following gradient
was used: 0–1 Min elution with 98% buffer, 1–20 Min gradually
decreased to 70% buffer, 20–23 Min quickly increased to 98%
buffer, and 23–30 Min elution with 98% buffer. The flow rate
was set to be 1.0 mL/Min. The injected volume was 20 µL.
The detection wavelength was 220 nm. The retention times
of the components were 2.4 Min for PG, 3.5 Min for 7-ADCA,
13.8 Min for PGME, and 17.0 Min for CEX. The concentrations
were calculated using calibration curves. All experiments were
performed in triplicate.
4 Efficient Enzymatic Synthesis of Cephalexin
Biotechnology and
Applied Biochemistry
3. Results and Discussion
3.1. Selection of addition manner of PGME
The solubility of PGME in the aqueous solution is more than
1.65 mol/L at pH 6.5 and 15 ◦ C as previously determined,
which is significantly higher than that of 7-ADCA. When the
enzymatic synthesis is performed in the suspension aqueous
solution system, an excess of molar ratio of PGME over 7-ADCA
can be formed in the liquid phase. As a result, the hydrolysis
of PGME to PG will be increased and higher amounts of PG are
produced. PG is sparingly soluble in water under the current
reaction conditions, and a large generation of PG will make the
downstream operations for the recovery of CEX and biocatalyst
cumbersome [6]. Therefore, to reduce the hydrolysis of PGME
in the enzymatic synthesis of CEX, the impact of the addition
of PGME, that is batch mode and continuous feeding on the
enzymatic synthesis, was investigated.
The time course for enzymatic synthesis of CEX with
the batch mode of PGME is shown in Fig. 2A. At 0 H, the
total concentration of 7-ADCA in suspension solution was
378 mmol/L, in which 39 mmol/L of 7-ADCA was dissolved in
a liquid phase and most of the 7-ADCA was suspended in the
form of solid particles. PGME was completely dissolved in the
liquid phase, and its concentration reached up to 450 mmol/L.
Additionally, 7 mmol/L of PG was detected in suspension
solution, which may be derived from self-driven hydrolysis of
PGME. The enzymatic synthesis reaction was initiated with
the addition of biocatalysts. At 0.33 H, the total concentration
of CEX was 153 mmol/L whereas it was 124 mmol/L in the
liquid phase. This illustrated that the concentration of CEX has
reached saturation at 0.33 H and its precipitation took place
in the suspension solution. At 0.67 H, PG started to precipitate
from the reaction mixtures and its subsequent decrease in
the liquid phase was observed. When the enzymatic synthesis
proceeded to 1.67 H, the maximum concentration of CEX
(315 mmol/L) was achieved. Meanwhile, residual 7-ADCA was
completely dissolved in the liquid phase due to its conversion
to CEX and its concentration was 29 mmol/L.
Figure 2B shows the time course for enzymatic synthesis
of CEX with the continuous feeding of PGME. At 0 H, the
total concentration of 7-ADCA in suspension solution was
530 mmol/L, in which 29 mmol/L of 7-ADCA was dissolved in the
liquid phase and most of the 7-ADCA was suspended in the form
of solid particles. The lower initial concentration of 7-ADCA in
the liquid phase in this case than that in the batch mode was due
to the increasing solubility of 7-ADCA with increasing amount
of PGME. A similar effect was reported about the solubility of
6-APA in the presence of PGME [39]. The enzymatic synthesis
reaction was initiated after the PGME solution was added into
the 7-ADCA suspension solution at a speed of 1 mL/Min with
a feeding time of 1 H. At 0.33 H, the concentration of CEX
in the liquid phase reached a maximum of 106 mmol/L and
then declined. Meanwhile, the total concentration of CEX was
106 mmol/L and increased afterward. These results suggested
that CEX started to precipitate from the reaction mixtures at
0.33 H. It should be noted that the maximum concentration of
CEX in the liquid phase with the continuous feeding was 14.9%
less than that with the batch mode. The concentrations of PGME
with the batch mode and continuous feeding at 0.33 H were 269
and 80 mmol/L, respectively. Obviously, the PGME concentration
in the former was 3.34 times of that of the latter. At the end
of the continuous feeding of PGME (1 H), the concentration
of PG in the liquid phase reached saturation and also started
to precipitate from the reaction mixture. It should be pointed
out that the time when PG precipitations appeared with the
continuous feeding of PGME was 0.33 H later than that with the
batch mode of PGME. This result indicated that the continuous
feeding of PGME could reduce the hydrolysis of PGME in the
enzymatic synthesis of CEX. When the enzymatic synthesis
reaction proceeded to 1.67 H, the total concentration of CEX in
the suspension aqueous solution reached the maximum value
of 325 and 16 mmol/L of unconverted 7-ADCA remained in
the liquid phase. No residual 7-ADCA was detected in the CEX
precipitation. As the enzymatic synthesis reaction was carried
on further, the concentration of 7-ADCA in the liquid phase
started to increase. The result indicated that the hydrolysis of
CEX to 7-ADCA and PG started to prevail over the synthesis of
CEX at this point.
Direct comparison of the performance for the enzymatic
synthesis with the batch mode and continuous feeding of
PGME is presented in Fig. 2C. For the continuous feeding, the
maximum conversion ratio of 7-ADCA, the productivity, and
the hydrolysis ratio of PGME was 96.3%, 194.3 mmol/L/H, and
15.4%, respectively. Compared to the case of batch mode, the
hydrolysis of PGME was reduced by 2.1%, while the conversion
ratio of 7-ADCA and productivity were improved by 2.6% and
2.7%, respectively. Therefore, the continuous feeding of PGME
is selected as a preferable strategy for enzymatic synthesis of
CEX in the suspension aqueous solution system.

3.2. Effect of process variables on the enzymatic

synthesis
As could be seen from Fig. 1, the kinetically controlled en-
zymatic synthesis of CEX was complex, which becomes even
more complicated in the suspension solution system due to
two additional processes, that is dissolution of 7-ADCA and
FIG. 2
precipitation (or crystallization) of CEX or PG. These processes
could be significantly affected by the reaction conditions, thus
affecting the efficiency of the whole enzymatic synthesis. There-
fore, the effect of various process variables must be studied to
improve the efficiency of the enzymatic synthesis.
3.2.1. Effect of pH and temperature
Because 7-ADCA is an amphoteric compound, its solubility in
aqueous solution strongly depends on the pH and temperature
of the reaction system. Moreover, the pH and temperature have
a significant influence on the catalytic activity of IPGA [40–42].
Therefore, the effect of pH and temperature on enzymatic
synthesis of CEX in the suspension aqueous solution was
investigated, and results are shown in Fig. 3.
(A) Concentration of 7-ADCA, PGME, CEX, and PG
in suspension aqueous solution with the batch
mode of PGME and (B) with continuous feeding of
PGME. The dashed line represents the
concentration of 7-ADCA in the liquid phase; the
short-dashed line represents the concentration of
CEX in the liquid phase; the dash-dotted line
represents the concentration of PG in the liquid
phase. (C) Performances of enzymatic synthesis of
CEX with two feeding manners of PGME. Reaction
conditions for (A): pH 6.5, 15 ◦ C, initial 7-ADCA 378
mmol/L, PGME/7-ADCA molar ratio 1.12,
IPGA/7-ADCA 30.46 IU/mmol, initial volume 216
mL, and final volume 242 mL; for (B): pH 6.5, 15 ◦ C,
initial 7-ADCA 531 mmol/L, PGME/7-ADCA molar
ratio 1.12, IPGA/7-ADCA 30.46 IU/mmol, PGME
feeding time 1 H, initial volume 154 mL, and final
volume 242 mL.

Biotechnology and Applied Biochemistry 5


Effect of pH (A) and temperature (B) on the
FIG. 3 enzymatic synthesis of CEX. Reaction conditions
for (A): 15 ◦ C, initial 7-ADCA 531 mmol/L,
PGME/7-ADCA molar ratio 1.12, IPGA/7-ADCA
30.46 IU/mmol, PGME continuous feeding time 1
H, initial volume 154 mL, and final volume 242 mL;
reaction conditions for (B): pH 7.0, initial 7-ADCA
531 mmol/L, PGME/7-ADCA molar ratio 1.12,
IPGA/7-ADCA 30.46 IU/mmol, PGME continuous
feeding time 1 H, initial volume 154 mL, and final
volume 242 mL.
Figure 3A shows the effect of pH on the enzymatic synthesis
of CEX. As shown in Fig. 3A1, within the range of pH 6.5–7.3,
the conversion ratio of 7-ADCA and productivity exhibited a
trend of increasing first and then decreasing. At pH 7.0, the
maximum conversion ratio and productivity of 98.8% and
250.6 mmol/L/H were achieved, respectively. In contrast, the
hydrolysis ratio of PGME showed an opposite trend, with
a minimum hydrolysis ratio of 12.0% at pH 7.0 (Fig. 3A2).
Interestingly, PG precipitation was detected in the reaction
system at pH 6.5 and 6.8, whereas no PG precipitation was
detected at pH 7.0. The results indicated that the hydrolysis of
PGME could be reduced at neutral condition at pH 7.0, resulting
in an increasing shift of PGME to the synthesis of CEX and
consequently elevating the conversion ratio and productivity. It
could also be seen from Fig. 3A2 that the solubility of 7-ADCA
increased and the reaction time for the maximum conversion
ratio of 7-ADCA declined with the increase of pH. However,
6 Efficient Enzymatic Synthesis of Cephalexin
Biotechnology and
Applied Biochemistry
when the pH was higher than 7.0, the hydrolysis ratio of
PGME started to increase. More PGME was consumed due
to the hydrolysis of PGME, which lead to the decrease in the
conversion ratio and productivity of enzymatic synthesis of
CEX. Clearly, the dual effects of increased solubility of 7-ADCA
and reaction rate at pH 7.0 led to the enhanced conversion ratio
and productivity and pH 7.0 was thus selected as the optimal
reaction pH.
Figure 3B shows the effect of temperature on the enzy-
matic synthesis of CEX at pH 7.0. As shown in Fig. 3B1, the
conversion ratio slightly increased with the temperature and
then decreased upon the temperature exceeding 15 ◦ C. The
maximum conversion ratio of 99.1% was obtained at 15 ◦ C. A
similar trend was observed for the productivity except that the
maximum productivity of 280 mmol/L/H was obtained at 20 ◦ C.
The solubility of 7-ADCA maintained a relatively stable level
(68–80 mmol/L) within the tested temperature range, whereas
the reaction time for the maximum conversion ratio declined
as the temperature increased (Fig. 3B2). Moreover, elevated
temperature enhanced the hydrolysis ratio of PGME. These
results suggested that increased temperature could improve
the catalytic activity of IPGA and accelerate the reaction rate,
and presumably the resultant productivity. However, the pro-
ductivity was affected by the hydrolysis ratio of PGME at a
higher temperature. If the conversion ratio of 7-ADCA was low,
the unreacted 7-ADCA was high in the solution and must be
recovered when the reaction was completed. The recovery of
unreacted 7-ADCA was difficult due to the coexisting of PG and
 Effect of enzyme loading (A) and feeding time of
FIG. 4
PGME (B) on the enzymatic synthesis of CEX.
Reaction conditions for (A): pH 7.0, 15 ◦ C, initial
7-ADCA 531 mmol/L, PGME/7-ADCA molar ratio
1.12, PGME continuous feeding time 1 H, initial
volume 154 mL, final volume 242 mL; reaction
conditions for (B): pH 7.0, 15 ◦ C, initial 7-ADCA 531
mmol/L, PGME/7-ADCA molar ratio 1.12,
IPGA/7-ADCA 30.46 IU/mmol, initial volume 154
mL, and final volume 242 mL..
CEX in the solution, which made the downstream processing
quite complicated. Therefore, in a trade-off between conversion
ratio and productivity, the optimal temperature was selected
as 15 ◦ C in subsequent experiments because the highest con-
version ratio was obtained at this condition with relatively high
productivity.
3.2.2. Effect of enzyme loading and feeding time of
PGME
Under the optimized pH and temperature, the effect of enzyme
loading on the enzymatic synthesis of CEX was investigated
and results are shown in Fig. 4A. The conversion ratio of 7-
ADCA was over 97% within the range of the enzyme loading
(15.23–38.08 IU/mmol). The maximum conversion ratio of
99.0% was obtained at the enzyme loading of 22.85 IU/mmol.
Additionally, the productivity exhibited a continuous increase
Biotechnology and Applied Biochemistry
with an increase in enzyme loading. Meanwhile, the minimum
hydrolysis ratio of 10.8% was also obtained at the enzyme
loading of 22.85 IU/mmol (Fig. 4A2). Higher enzyme loading,
for example, 38.08 IU/mmol resulted in PG precipitation in
the solution. The low enzyme loading extended the reaction
time for the maximum conversion ratio, explaining that the
hydrolysis of PGME was exacerbated and the conversion of
7-ADCA was reduced. The high enzyme loading could enhance
the hydrolytic activity of biocatalyst, which would lead to
increased hydrolysis of PGME and decreased conversion of
7-ADCA. As shown in Fig. 4A2, the reaction time for maximum
conversion of 7-ADCA significantly decreased with the increase
of enzyme loading, and thus resulted in the improvement of
productivity. Considering that the specific productivity (the
CEX mmols produced per unit time and unit of biocatalysts
mass at maximum conversion) was 3.30 mmol/H/gcat under the
enzyme loading of 22.85 IU/mmol, which was higher than that
(2.94 mmol/H/gcat ) under the enzyme loading of 30.46 IU/mmol,
the optimal enzyme loading was selected as 22.85 IU/mmol for
further experiments.
The continuous feeding method of PGME was beneficial for
enzymatic synthesis of CEX as described above. The feeding
time of PGME relating to the concentration of PGME in solution
and its hydrolysis ratio may have an impact on the conversion
ratio and productivity. Therefore, the effect of feeding time on
7

Effect of the initial concentration of 7-ADCA (A)
FIG. 5 and PGME/7-ADCA molar ratio (B) on the
enzymatic synthesis of CEX. Reaction conditions
for (A): pH 7.0, 15 ◦ C, PGME/7-ADCA molar ratio
1.12, IPGA/7-ADCA 22.85 IU/mmol, and PGME
continuous feeding time 1 H; reaction conditions
for (B): pH 7.0, 15 ◦ C, initial 7-ADCA 659 mmol/L,
IPGA/7-ADCA 30.46 IU/mmol, PGME continuous
feeding time 1 H, initial volume 124 mL, and final
volume 210 mL.
the enzymatic synthesis of CEX was investigated. As shown in
Fig. 4B1, the maximum conversion ratio of 99.2% was reached
at the feeding time of 1 H. When the feeding time was more
than 1 H, the conversion ratio of 7-ADCA showed a slight
decrease. In contrast, the productivity exhibited a decreasing
tendency with feeding time. As shown in Fig. 4B2, the reaction
time for maximum conversion of 7-ADCA was extended with
an increase of feeding time, indicating that the reaction rate
for CEX synthesis decreased. As a result, the productivity
was decreased. Additionally, the hydrolysis ratio of PGME
slowly declined with the increase of feeding time, indicating
that increasing the feeding time of PGME could reduce its
hydrolysis, thus enhancing the conversion ratio of 7-ADCA
and the production of CEX. As a result, 1 H of feeding time is
suitable for the maximum conversion ratio of 7-ADCA.
8 Efficient Enzymatic Synthesis of Cephalexin
Biotechnology and
Applied Biochemistry
3.2.3. Effect of the initial concentration of 7-ADCA and
PGME/7-ADCA molar ratio
The initial concentration of 7-ADCA in the suspension solu-
tion has a direct influence on the mass transfer as well as
the PGME hydrolysis and reaction productivity of the enzy-
matic synthesis of CEX. Figure 5A shows the effect of initial
7-ADCA concentration on the enzymatic synthesis of CEX. As
shown in Fig. 5A1, as the initial concentration of 7-ADCA in-
creased, both of the conversion ratio and productivity showed
increasing tendency within the range of 532–746 mmol/L.
The maximum conversion ratio of 99.7% and productivity of
214.6 mmol/L/H were obtained at the 7-ADCA concentration of
746 mmoL/L. The reaction time under different initial 7-ADCA
concentrations did not show a significant change (Fig. 5A2).
However, the hydrolysis ratio of PGME declined significantly
with an increase of the initial 7-ADCA concentration. The
minimum hydrolysis ratio of 10.8% was achieved when the
initial concentration of 7-ADCA was increased to 746 mmoL/L.
Reducing the water activity of aqueous medium has been
known to inhibit the hydrolysis of PGME in the enzymatic
synthesis of CEX [15,16]. Therefore, it was concluded that the
increasing initial concentration of 7-ADCA decreased the water
activity of the reaction system and decreased the hydrolysis
ratio of PGME. Accordingly, more PGME and 7-ADCA would
be transferred to synthesize of CEX. Additionally, it was found

(A) Time course of the concentration of 7-ADCA,
FIG. 6 PGME, CEX, and PG in the suspension aqueous
solution system and (B) homogeneous aqueous
solution system. The sashed line represents the
concentration of 7-ADCA in the liquid phase; the
short-dashed line represents the concentration of
CEX in the liquid phase. Reaction conditions for
(A): pH 7.0, 15 ◦ C, initial 7-ADCA 659 mmol/L,
PGME/7-ADCA molar ratio 1.12, IPGA/7-ADCA
22.85 IU/mmol, PGME continuous feeding time
1 H, initial volume 124 mL, and final volume 210
mL; for (B): pH 7.0, 15 ◦ C, initial 7-ADCA 100
mmol/L, PGME/7-ADCA molar ratio 1.12,
IPGA/7-ADCA 22.85 IU/mmol, initial and final
volume 817 mL.
that the concentration of PG in the aqueous solution increased
with the increase of concentration of 7-ADCA. Although higher
conversion ratio and productivity of CEX were obtained at
746 mmol/L of the initial concentration of 7-ADCA, PG pre-
cipitation was also found in the precipitation of CEX, making
downstream operation difficult for the recovery of CEX. In con-
trast, no precipitation of PG was detected in the reaction system
at the 7-ADCA concentration of 659 mmol/L. Meanwhile, the
conversion ratio and productivity still reached to 99.4% and
199.8 mmol/L/H, respectively. Therefore, the initial 7-ADCA
Biotechnology and Applied Biochemistry
concentration of 659 mmol/L was selected at the preferred
condition for further experiments.
High PGME/7-ADCA molar ratio is the often applied to
achieve a high conversion ratio of 7-ADCA to CEX. However,
excessive consumption of PGME would result in an increase
in production costs and waste discharge as stated in the In-
troduction section. Under the above optimal conditions, the
effect of PGME/7-ADCA molar ratio on the enzymatic synthesis
of CEX was investigated and results are shown in Fig. 5B. It
was observed that the conversion ratio of 7-ADCA appeared
to show a continuous rise as the PGME/7-ADCA molar ratio
increased within the range of 1.04–1.16 (Fig. 5B1). The max-
imum conversion ratio of 99.7% was obtained at the molar
ratio of 1.16. The productivity also increased concomitantly. As
shown in Fig. 5B2, the reaction time at different PGME/7-ADCA
molar ratios did also not show a significant change, whereas
the hydrolysis ratio of PGME increased monotonously with the
increase of PGME/7-ADCA molar ratio. Especially, PG precip-
itation was detected in the reaction system at the molar ratio
of 1.16. The results indicated that the increased PGME/7-ADCA
molar ratio was beneficial for enhancing the conversion ratio
of 7-ADCA, but it also increased the hydrolysis ratio of PGME
and generated large amounts of PG that was disadvantageous
to downstream separation and purification. No PG precipitation
was determined in the reaction system at the PGME/7-ADCA
molar ratio of 1.12, and the corresponding conversion ratio and
productivity were 99.3% and 200 mmol/L/H, respectively. By
comprehensive consideration, the PGME/7-ADCA molar ratio
of 1.12 was selected as the best condition in terms of its almost
stoichiometric conversion ratio at high values of productivity
as well as lower amounts of PG. To the best of our knowledge,
this is the lowest PGME/7-ADCA molar ratio that can be used
for enzymatic synthesis of CEX with high conversion ratio and
productivity.
3.3. Performance of the developed approach
Through investigating the process parameters above, the
optimal conditions for enzymatic synthesis of CEX by the
manner of continuous feeding of PGME was achieved: pH 7.0,
15 ◦ C, initial 7-ADCA 659 mmol/L, PGME/7-ADCA molar ratio
1.12, IPGA/7-ADCA 22.85 IU/mmol, and PGME continuous
feeding time 1 H. To further evaluate the developed approach,
the variation of concentration of the reactants, product CEX,
and by-product PG under the optimized reaction conditions was
investigated. The time course for enzymatic synthesis of CEX
in the suspension aqueous solution system is shown in Fig. 6A.
When the reaction proceeded to 0.33 H, the concentration of
CEX in the liquid phase reached a maximum of 132 mmol/L,
and then precipitation of CEX took place. When the reaction
time was extended over 1 H, the total concentration of 7-ADCA
and that in the liquid phase was overlapped, indicating that
7-ADCA was completely dissolved in the aqueous solution. The
minimum concentration (5 mmol/L) of 7-ADCA was observed
at 2 H, corresponding to the maximum concentration of CEX,
which indicated that the maximum conversion of 7-ADCA to
9
 Biotechnology and
Applied Biochemistry

Comparison for enzymatic synthesis of CEX in the suspension aqueous solution system and homogeneous aqueous solution
TABLE 1 system

Initial concentrations of
reactants Performance under the optimum conditions
a
7-ADCA PGME
Reaction system (mmol/L) (mmol/L) CR (%) P (mmol/L/H) HR (%)

Suspension aqueous 659 – 99.3 200 11.4

b
solution
c
Homogeneous aqueous 100 112 94.2 40.4 18.0
solution
a The concentration of PGME in the stock solution was 1.65 mol/L. However, the PGME was added into the tank by a continuous feeding manner;

Therefore we could not put a value for the initial concentration in the grid of the table.
b Reactionconditions: pH 7.0, 15 ◦ C, initial 7-ADCA 659 mmol/L, PGME/7-ADCA molar ratio 1.12, IPGA/7-ADCA 22.85 IU/mmol, PGME continuous
feeding time 1 H, total reaction time 2 H, initial volume 124 mL, and final volume 210 mL.
c Reaction conditions: pH 7.0, 15 ◦ C, initial 7-ADCA 100 mmol/L, PGME/7-ADCA molar ratio 1.12, IPGA/7-ADCA 22.85 IU/mmol, total reaction time
2.33 H, the initial and final volume 817 mL.

its entire process consists of five related subprocesses: enzy-

Recycling stability of IPGA for the synthesis of CEX
FIG. 7 in the suspension aqueous solution system.
Reaction conditions: pH 7.0, 15 ◦ C, initial 7-ADCA
659 mmol/L, PGME/7-ADCA molar ratio 1.12,
IPGA/7-ADCA 22.85 IU/mmol, PGME continuous
feeding time 1 H, total reaction time 2 H, initial
volume 124 mL, and final volume 210 mL.
CEX was achieved. At the time point, the reaction was once
terminated and the reaction mixtures were filtrated. The
compositions of the precipitated product and the filtrate were
separately analyzed by HPLC. It was interesting to find that
only trivial amount of PG was present in the obtained CEX
precipitations, which was much lower than that in the filtrate
(Fig. S2 in the Supporting Formation). Through calculation, the
contents of CEX and PG in the precipitated product were 99.5%
(w/w) and 0.5% (w/w), respectively. These results exhibited the
great advantage of the developed approach.
Unlike the traditional aqueous solution system, the sus-
pension aqueous solution system was a more complex reaction
system for enzymatic synthesis of CEX. As shown in Fig. 1B,
matic synthesis of CEX(1), enzymatic hydrolysis of PGME(2),
enzymatic hydrolysis of CEX(3), dissolution of 7-ADCA(4), and
precipitation of CEX (5). The enzymatic synthesis of CEX in
the suspension aqueous solution was largely controlled by the
enzymatic hydrolysis of PGME and dissolution of 7-ADCA. First,
there existed a competition between the enzymatic synthesis of
CEX and enzymatic hydrolysis of PGME. Second, according to
the Michaelis–Menten equation, the conversion ratio of 7-ADCA
would be increased by means of the increase of its concentra-
tion in the liquid phase, which was equal to its solubility in this
case [43]. As a result, an increase in solubility would increase
the conversion ratio of 7-ADCA. Otherwise, a decrease in solu-
bility would decrease the conversion ratio of 7-ADCA, and the
hydrolysis of PGME will be increased. This is in agreement with
the results shown in Fig. 3A. Additionally, the precipitation of
CEX has the advantage of reducing the ratios of CEX hydrolysis
because CEX is protected from the enzyme whenever it is in
the solid phase, which was also beneficial for the enzymatic
synthesis of CEX.
To make a comparison, the time course of enzymatic
synthesis of CEX in the homogeneous aqueous solution was
also studied, where the initial feeding concentration of 7-ADCA
was 100 mmol/L, which was close to its limit of solubility under
the reaction condition. As shown in Fig. 6B, when IPGA was
added into the reaction system, the concentration of 7-ADCA
rapidly decreased and the concentration of CEX increased
correspondingly. Meanwhile, the concentration of PG started
to slowly increase. When the reaction proceeded to 2.33 H,
the minimum 4 mmol/L of 7-ADCA and maximum 94 mmol/L
of CEX were both achieved. During the reaction, CEX was
completely dissolved in the aqueous solution.
Direct comparisons of the performances for both
systems are presented in Table 1. In suspension aqueous

10 Efficient Enzymatic Synthesis of Cephalexin

solution system, the hydrolysis ratio of PGME was 11.4%, a
conversion ratio of 7-ADCA was 99.3%, and productivity was
200 mmol/L/H. In the aqueous solution system, the hydrolysis
of PGME was 18.0%, the conversion ratio of 7-ADCA was
94.2%, and productivity was 40.4 mmol/L/H. It is clear that the
hydrolysis ratio of PGME was decreased by 6.6%, the conver-
sion ratio of 7-ADCA was increased by 5.1%, and productivity
was increased by 395% from the homogeneous solution system
to the suspension solution system. These results demonstrated
that the suspension aqueous solution system exhibited superior
performance for enzymatic synthesis of CEX.
3.4. Recycling stability of IPGA for the synthesis of
CEX in suspension aqueous solution system
The stability of IPGA is crucial to the cost of industrial pro-
duction of CEX by an enzymatic route. To further evaluate
the potential of the developed approach for industrial produc-
tion, the recycling of the IPGA for the synthesis of CEX in the
suspension aqueous system was investigated by measuring
the conversion ratio and productivity under the optimized
conditions. As shown in Fig. 7, in the first batch, the conver-
sion ratio and productivity were 99.3% and 200 mmol/L/H,
respectively. After 10 runs, the conversion ratio is still up to
94.7% with productivity of 189.8 mmol/L/H. In other words,
the IPGA still maintained 95.4% of its original activity, re-
flecting excellent stability of IPGA in the suspension aqueous
solution.
4. Conclusions
In this work, an efficient approach was successfully developed
for the enzymatic synthesis of CEX in the suspension aqueous
solution system by continuous feeding of PGME. The maximal
conversion ratio of 99.3% for 7-ADCA and productivity of
200 mmol/L/H were achieved at a PGME/7-ADCA molar ratio of
1.12. Compared with the homogenous aqueous solution system,
this approach exhibited advantages of high initial feeding
concentration of 7-ADCA, conversion ratio, and productivity.
Recycling of enzyme in the experiment demonstrated good
stability of IPGA. The work would be helpful in the industrial
production of CEX by the enzymatic route.
5. Acknowledgements
The authors gratefully acknowledge financial support from
Major Science and Technology Project of Water Pollution
Control and Management in China (2017ZX07402003), the Key
Research Program of the Chinese Academy of Sciences (ZDRW-
ZS-2016-5-3) and the National Natural Science Foundation of
China (21676272).
6. References
[1] Wegman, M. A., Janssen, M. H., Van Rantwijk, F., and Sheldon, R. A. (2001)
Adv. Synth. Catal. 343, 559–576.
[2] Elander, R. P. (2003) Appl. Microbiol. Biotechnol. 61, 385–392.
Biotechnology and Applied Biochemistry
[3] Giordano, R. C., Ribeiro, M. P.,and Giordano, R. L. (2006) Biotechnol. Adv. 24,
27–41.
[4] Cole, M. (1969) Biochem. J. 115, 747–756.
[5] Mateo, C., Abian, O., Grazú, V., Fernández-Lorente, G., Palomo, J. M., Fuentes,
M., Rosa, L. S., Tamara, M., Fernando, L. G., Lorena, W., Rodrigo, T., José, M.
G., and Torres, R. (2005) Med. Chem. Rev. 2, 207–218.
[6] Encarnación-Gómez, L. G., Bommarius A. S., and Rousseau R. W. (2006)
React. Chem. Eng. 1, 321–329.
[7] Bruggink, A., Roos, E. C., and De Vroom, E. (1998) Org. Process Res. Dev. 2,
228-133.
[8] McDonald, M. A., Bommarius, A. S., Rousseau, R. W., and Grover, M. A.
(2019) Comput. Chem. Eng. 123, 331–343.
[9] Illanes, A., Cabrera, Z., and Wilson, L., Aguirre, C. (2003) Process Biochem.
39, 111–117.
[10] Illanes, A., Anjari, M. S., Altamirano, C., and Aguirre, C. (2004) J. Mol. Catal.
B Enzyme 30, 95–103.
[11] Aguirre, C., Opazo, P., Venegas, M., Riveros, R., and Illanes, A. (2006) Process
Biochem. 41, 1924–1931.
[12] Van Langen, L. M., De Vroom, E., Van Rantwijk, F., and Sheldon, R. (1999)
FEBS Lett. 456, 89–92.
[13] Aguirre, C., Toledo, M., and Medina, V., Illanes, A. (2002) Process Biochem.
38, 351–360.
[14] Illanes, A., Anjarı́, S., Arrieta, R., and Aguirre, C. (2002) Appl. Biochem.
Biotechnol. 97, 165–179.
[15] Hyun, C. K., Choi, J. H., Kim, J. H., and Ryu, D. D. (1993) Biotechnol. Bioeng.
41, 654–658.
[16] Hyun, C. K., Kim, J. H., and Ryu, D. D. (1993) Biotechnol. Bioeng. 42, 800–806.
[17] Li, S., and Cao, X. (2014) Biochem. Eng. J. 90, 301–306.
[18] Aguirre, C., Concha, I., Vergara, J., Riveros, R., and Illanes, A. (2010) Process
Biochem. 45, 1163–1167.
[19] Cao, X., Zhu, J., Wei, D., and Hur, B. K. (2004) J. Microbiol. Biotechnol. 14,
62–67.
[20] Wei, D. Z., Zhu, J. H., and Cao, X. J. (2002) Biochem. Eng. J. 11, 95–99.
[21] Li, D., Zhang, Y., Cheng, S., Gao, Q., and Wei, D. (2008) Food Technol.
Biotechnol. 46, 461–466.
[22] Schroën, C. G. P. H., Nierstrasz, V. A., Bosma, R., Kemperman, G. J., Strubel,
M., Ooijkaas, L. P., Beeftink, H. H., and Tramper, J. (2002) Enzyme Microb.
Technol. 31, 264–273.
[23] Zhou, H. C., Li, W., Shou, Q. H., Gao, H. S., Xu, P., Deng, F. L., and Liu, H. Z.
(2012) Chin. J. Chem. Eng. 20, 146–151.
[24] Pan, X., Wang, L., Ye J., Qin, S., and He, B. (2018) Appl. Microbiol. Biotechnol.
102, 1749–1758.
[25] Bernardino, S. M. S. A., Fernandes, P., and Fonseca, L. P. (2010) Biotechnol.
Bioeng. 107, 753–762.
[26] Basso, A., Spizzo, P., Toniutti, M., Ebert, C., Linda, P., and Gardossi, L. (2006)
J. Mol. Catal. B: Enzyme 39, 105–111.
[27] Bahamondes, C., Wilson, L., Aguirre, C., and Illanes, A. (2012) Biotechnol.
Bioprocess. E. 17, 711–721.
[28] Illanes, A., Altamirano, C., Fuentes, M., Zamorano, F., and Aguirre, C. (2005)
J. Mol. Catal. B: Enzyme 35, 45–51.
[29] Illanes, A., Wilson, L., Altamirano, C., Cabrera, Z., Alvarez, L., and Aguirre, C.
(2007) Enzyme Microb. Technol. 40, 195–203.
[30] Illanes, A., Wilson, L., Corrotea, O., Tavernini, L., Zamorano, F., and Aguirre,
C. (2007) J. Mol. Catal. B: Enzyme 47, 72–78.
[31] Illanes, A., Wilson, L., and Aguirre, C. (2009) Appl. Biochem. Biotechnol. 157,
98–110.
[32] Youshko, M. I., Moody, H. M., Bukhanov, A. L., Boosten, W. H., and Švedas,
V. K. (2004) Biotechnol. Bioeng. 85, 323–329.
[33] Chaiwut, P., Kanasawud, P., and Halling, P. J. (2007) Enzyme Microb. Technol.
40, 954–960.
[34] Lee, D. C., and Kim, H. S. (1998) Biotechnol. Bioeng. 60, 729–738.
[35] Erbeldinger, M., Ni, X., and Halling, P. J. (2001) Biotechnol. Bioeng. 72, 69–
76.
[36] Ulijn, R. V., Janssen, A. E., Moore, B. D., and Halling, P. J. (2001) Chem-Eur. J.
7, 2089–2098.
11

[37] Ulijn, R. V., Martin, L. D., Gardossi, L., and Halling, P. J. (2003) Curr. Org.
Chem. 7, 1333–1346.
[38] Erbeldinger, M., Ni, X., and Halling, P. J. (1998) Enzyme Microb. Technol. 23,
141–148.
[39] Youshko, M. I., Van Langen, L. M., De Vroom, E., Moody, H. M., Van Rantwijk
F., Sheldon, R. A., and Švedas, V. K. (2000) J. Mol. Catal. B: Enzyme 10,
509–515.
12
Biotechnology and
Applied Biochemistry
[40] Illanes, A., and Fajardo, A. (2001) J. Mol. Catal. B: Enzyme 11, 587–595.
[41] Ospina, S., Barzana, E., Ramı́rez, O. T., and López-Munguı́a A. (1996) Enzyme
Microb. Technol. 19, 462–469.
[42] Youshko M. I., Van Langen L.M., De Vroom E., Van Rantwijk F., Sheldon R. A.,
and Švedas V.K. (2002) Biotechnol. Bioeng. 78, 589–593.
[43] Wolff, A., Zhu, L., Kielland, V., Straathof, A. J. J., Jongejan, J. A., and Heijnen,
J. J. (1997) Biotechnol. Bioeng. 56, 433–440.
Efficient Enzymatic Synthesis of Cephalexin
Overall reaction scheme involved in the
enzymatic synthesis of cephalexin in
suspension aqueous solution system with
precipitated substrate 7-ADCA and
product CEX.
Page 1–12
Yixiao Fan, Yingbo Li, Qingfen Liu
Efficient enzymatic synthesis of cephalexin in
suspension aqueous solution system