Kinetically controlled acylation of 6-APA catalyzed by penicillin acylase from Streptomyces lavendulae: effect of reaction conditions in the enzymatic synthesis of penicillin V

Article abstract of DOI:10.1080/10242422.2019.1652274

Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin acylase from Streptomyces lavendulae (SlPVA) as biocatalyst. The effect of different reaction conditions on penV synthesis was investigated, such as enzyme concentration, pH, molar ratio of 6-APA to MPOA, as well as presence of DMSO as water-miscible co-solvent at different concentrations. Time-course profiles of all reactions followed the typical pattern of kinetically controlled synthesis (KCS) of β-lactam antibiotics: penV concentration reached a maximum (highest yield or Y_max) and then decreased gradually. Such maximum was higher at pH 7.0, observing that final penV concentration was abruptly reduced when basic pH values were employed in the reaction. Under the selected conditions (100 mM Tris/HCl buffer pH 7.0, 30 °C, 2.7percent (v/v) DMSO, 20 mM MPOA, 0.3 UI/ml of SlPVA), Y_max was enhanced by increasing the substrate molar ratio (6-APA to MPOA) up to 5, reaching a maximum of 94.5percent and a S/H value of 16.4 (ratio of synthetic activity to hydrolytic activity). As a consequence, the use of an excess of 6-APA as nucleophile has allowed us to obtain some of the highest Y_max and S/H values among those reported in literature for KCS of β-lactam antibiotics. Although many penicillin G acylases (PGAs) have been described in kinetically controlled acylations, SlPVA should be considered as a different enzyme in the biocatalytic tool-box for novel potential synthetic processes, mainly due to its different substrate specificity compared to PGAs.

Full text of DOI:10.1080/10242422.2019.1652274

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20
Kinetically controlled acylation of 6-APA catalyzed
by penicillin acylase from Streptomyces lavendulae:
effect of reaction conditions in the enzymatic
synthesis of penicillin V
Daniel Hormigo, María Teresa López-Conejo, Lara Serrano-Aguirre, Alberto
García-Martín, Ana Saborido, Isabel de la Mata & Miguel Arroyo
To cite this article: Daniel Hormigo, María Teresa López-Conejo, Lara Serrano-Aguirre, Alberto
García-Martín, Ana Saborido, Isabel de la Mata & Miguel Arroyo (2019): Kinetically controlled
acylation of 6-APA catalyzed by penicillin acylase from Streptomycesꢀlavendulae: effect of reaction
conditions in the enzymatic synthesis of penicillin V, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2019.1652274
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2019.1652274
RESEARCH ARTICLE
Kinetically controlled acylation of 6-APA catalyzed by penicillin acylase
from Streptomyces lavendulae: effect of reaction conditions in the enzymatic
synthesis of penicillin V
ꢀ
Daniel Hormigo , Mar ꢀı a Teresa L oꢀ pez-Conejo, Lara Serrano-Aguirre, Alberto Garc ꢀı a-Mart ꢀı n, Ana Saborido,
Isabel de la Mata and Miguel Arroyo
Department of Biochemistry and Molecular Biology, Faculty of Biology, Enzyme Biotechnology Group, Universidad Complutense de
Madrid, Madrid, Spain
ABSTRACT
ARTICLE HISTORY
Received 22 May 2019
Revised 2 July 2019
Accepted 31 July 2019
Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was
carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin
acylase from Streptomyces lavendulae (SlPVA) as biocatalyst. The effect of different reaction con-
ditions on penV synthesis was investigated, such as enzyme concentration, pH, molar ratio of
KEYWORDS
Penicillin acylase;
Streptomyces lavendulae;
kinetic controlled synthesis;
acylation; penicillin V
6-APA to MPOA, as well as presence of DMSO as water-miscible co-solvent at different concen-
trations. Time-course profiles of all reactions followed the typical pattern of kinetically controlled
synthesis (KCS) of b-lactam antibiotics: penV concentration reached a maximum (highest yield or
Ymax) and then decreased gradually. Such maximum was higher at pH 7.0, observing that final
penV concentration was abruptly reduced when basic pH values were employed in the reaction.
ꢁ
Under the selected conditions (100 mM Tris/HCl buffer pH 7.0, 30 C, 2.7% (v/v) DMSO, 20 mM
MPOA, 0.3 UI/ml of SlPVA), Ymax was enhanced by increasing the substrate molar ratio (6-APA to
MPOA) up to 5, reaching a maximum of 94.5% and a S/H value of 16.4 (ratio of synthetic activity
to hydrolytic activity). As a consequence, the use of an excess of 6-APA as nucleophile has
allowed us to obtain some of the highest Ymax and S/H values among those reported in litera-
ture for KCS of b-lactam antibiotics. Although many penicillin G acylases (PGAs) have been
described in kinetically controlled acylations, SlPVA should be considered as a different enzyme
in the biocatalytic tool-box for novel potential synthetic processes, mainly due to its different
substrate specificity compared to PGAs.
1. Introduction
antibiotic (h ). On the one hand, the degree of satur-
2
ation of the enzyme active site by 6-APA is a deter-
mining factor in increasing the synthesis yield
Kinetically controlled synthesis (KCS) of b-lactam anti-
biotics using free or immobilized penicillin acylases
has been successfully employed for the production of
semisynthetic penicillins and cephalosporins (Arroyo
et al. 2003; Volpato et al. 2010; Sklyarenko et al. 2015).
This approach involves an acyl group transfer reaction
between a nucleophile (b-lactam nuclei such as 6-ami-
nopenicillanic acid (6-APA), 7-aminocephalosporanic
acid (7-ACA), or 7-aminodesacetoxycephalosporanic
acid (7-ADCA) and the corresponding activated acyl
donor (esters, amides or anhydrides of acids)
(
Gon c¸ alves, Fern ꢀa ndez-Lafuente, et al. 2002). In this
sense, antibiotic synthesis requires adsorption of 6-
APA on the enzyme active site before the substrate-
enzyme complex is formed (Kasche 1986). On the
other hand, reaction conditions must be carefully
chosen in order to favour the synthetic process and to
reduce the hydrolytic ones. This goal has been
achieved through the optimization of the reaction
conditions such as an appropriate pH value (Ospina
et al. 1996; Aguirre et al. 2002; Youshko, van Langen,
et al. 2002; Illanes et al. 2003), the addition of a suit-
able water-miscible organic co-solvent (Rosell et al.
1998; Illanes and Fajardo 2001; Aguirre et al. 2002;
Illanes et al. 2002; Wei and Yang 2003; Chow et al.
(Bruggink et al. 1998; Wegman et al. 2001). The overall
synthesis yield depends on the balance of the rates of
three different reactions catalyzed by the enzyme, not
only the antibiotic synthesis (S), but the acyl donor
hydrolysis (h ) and the hydrolysis of the synthesized
1
CONTACT Miguel Arroyo
arroyo@bio.ucm.es
Department of Biochemistry and Molecular Biology, Faculty of Biology, Enzyme Biotechnology
Group, Universidad Complutense de Madrid. c/Jos ꢀe Antonio Novais, 12. 28040, Madrid, Spain
ꢀ
Present address: Applied Biotechnology Group, European University of Madrid, Urbanizaci ꢀo n “El Bosque”, Tajo s/n, 28670 Villaviciosa de Od ꢀo n, Spain
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
D. HORMIGO ET AL.
2
007; Zhang and Wei 2008; Pereira et al. 2012), a high
industry (Mare ꢁs ov ꢀa et al. 2014). Despite their rele-
vance, other penicillin acylases such as penicillin V
acylases (PVAs) (Zhang et al. 2007; Kumar et al. 2008;
Avinash et al. 2015; Xu et al. 2018) or aliphatic penicil-
lin acylases (Torres-Guzm ꢀa n et al. 2002; Torres-Bacete
et al. 2007) should be taken into consideration in
order to widen the biocatalytic tool-box for synthetic
processes. As a matter of fact, PVA from Streptomyces
mobaraensis (SmPVA) (Koreishi et al. 2007), and PVA
from Erwinia aroideae (EaPVA) (Nam and Ryu 1984) are
the only members of the PVA family to efficiently cata-
lyse the synthesis of penicillin V (penV) using a KCS
process, and SmPVA was also able to synthesize other
b-lactam antibiotics (such as penicillins F and K, as
well as deacetoxycephalosporin V), capsaicin deriva-
tives, and N-fatty-acylated amino compounds (Koreishi
et al. 2007). Aliphatic penicillin acylase from
Streptomyces lavendulae (SlPVA) has been exhaustively
characterized (Arroyo et al. 1999, 2000; Torres-
Guzm ꢀa n, de la Mata, Arroyo, et al. 2001; Torres-
molar ratio of activated acyl donor to nucleophile
Illanes et al. 2007; Aguirre et al. 2010), an excess of
nucleophile concentration in the reaction medium
Chow et al. 2005), the application of homogeneous
supersaturated solutions of substrates (Youshko et al.
004; Illanes et al. 2007), or the use of aqueous two-
(
(
2
phase systems for in situ extraction of the reaction
products (Hern ꢀa ndez-J uꢀ stiz et al. 1998; Terreni et al.
2
approach has resulted equally well for the preparation
of a wide number of semisynthetic b-lactam antibiot-
ics (Mare ꢁs ov ꢀa et al. 2014). All of these synthetic reac-
tions have been catalyzed by penicillin G acylases
005; Aguirre et al. 2010). As a result, the KCS
(PGAs) (Giordano et al. 2006; Chandel et al. 2008), and
some of their kinetics have been systematically
studied (Youshko et al. 2003; Giordano et al. 2006).
Consequently, a few relevant parameters for the evalu-
ation of the synthetic process have been described.
Essentially, the activated acyl donor is attacked by Od
of bSer1 of the enzyme to form an acyl-enzyme inter-
mediate. This covalent intermediate can be either
hydrolyzed by water, yielding the free acid of the acyl
donor, or aminolyzed by a b-lactam nucleus, yielding
the desired antibiotic. The ratio of initial rate of anti-
Guzm ꢀa n,
de
la
Mata,
Torres-Bacete,
2001;
Torres-Guzm ꢀa n et al. 2002; Torres-Bacete et al. 2015)
due to its ability to deacylate not only penV but nat-
ural aliphatic penicillins (such as penicillin K, penicillin
dihydroF and penicillin F) to 6-APA. The aim of the
present work is to study the effect of reaction condi-
tions on the KCS of penV catalyzed by SlPVA. Effects
of enzyme concentration, pH, molar ratio of 6-APA to
acyl donor, and DMSO (as organic co-solvent at differ-
ent concentrations) on penV synthesis were studied
and discussed.
biotic synthesis (V ) to initial rate of free acid forma-
tion (V ) is known as the S/H ratio or selectivity
coefficient (Hern ꢀa ndez-J uꢀ stiz et al. 1999; Terreni et al.
S
h1
2007). Since no antibiotic is present at the beginning
of the reaction, initial rate of antibiotic hydrolysis (Vh2)
can be neglected in the initial phase of the conversion
(Youshko et al. 2003). Hence, S/H ratio indicates the
preference of the acyl-enzyme intermediate to interact
with the b-lactam nucleus instead of water (Youshko,
Chilov, et al. 2002). As the process advances, the
formed antibiotic can be also hydrolyzed by the peni-
cillin acylase, and antibiotic concentration will reach a
maximum (maximum yield, Ymax or [PS]max), after
which it will decrease again. Once Ymax is estimated,
maximum productivity (Pmax) can be determined as
the amount of antibiotic produced per unit of time at
maximum yield (Illanes et al. 2007, 2009). As rule of
thumb, S/H ratio has been used for the evaluation of
the synthetic potential of a selected penicillin acylase
2
. Materials and methods
2.1. Materials
penV (potassium salt), p-diaminobenzaldehyde (PDAB),
phenoxyacetic acid (POA) and methyl phenoxyacetate
(MPOA) were supplied by Sigma-Aldrich (St. Louis,
MO). 6-APA were provided by Antibioticos S.A. (Leon,
Spain). Tris, potassium phosphate, acetic acid and
other chemicals were supplied by Fisher Scientific
ꢀ
ꢀ
(Hampton, NH).
2.2. Penicillin acylase production
(soluble or immobilized, wild-type or mutant) (Cheng
et al. 2006; Jager et al. 2007, 2008; Estruch et al. 2008).
As mentioned before, PGAs from different microbial
sources (mainly from Escherichia coli, hereafter abbre-
viated as EcPGA) and their enzyme variants (mutant,
evolved or immobilized) have been the most common
biocatalysts used for the synthesis of b-lactam antibi-
otics, gaining a unique position in pharmaceutical
A culture recombinant Streptomyces lividans strain
overexpressing SlPVA (Torres-Bacete et al. 2015) was
grown aerobically under submerged conditions in
250-ml Erlenmeyer flasks containing 100 ml of Tryptic
Soy Broth (TSB) medium (Becton Dickinson, San Jose,
CA). Fermentation was carried out by inoculating
6
2 ꢂ 10 spores/ml of culture medium and using an

BIOCATALYSIS AND BIOTRANSFORMATION
3
ꢁ
incubation shaker at 250 rpm and 30 C for 96 h. Once
(POA), 8.3 min (MPOA) and 10.4 min (penV).
Concentrations of penV, POA and MPOA were quanti-
tatively estimated with the corresponding calibration
the cells were removed by centrifugation at 3000ꢂg,
the cloned enzyme was purified from the culture
broth in one step by ionic exchange chromatography
using S-sepharose from Amersham BioSciences
Buckinghamshire, UK). Specific activity of the pure
recombinant enzyme after its purification was
1.0 IU/mg by employing the spectrophotometric
assay for enzyme activity which is described below.
curves using standard solutions. Synthetic activity (V )
S
was established as the initial rate of synthesis (penV
production per unit time, expressed as mM/h),
whereas hydrolytic activity (Vh1) was established as the
initial rate of hydrolysis of the acyl donor MPOA (POA
production per unit time, expressed as mM/h). Since
no antibiotic is present at the beginning of the reac-
tion, penV hydrolysis can be neglected in the initial
phase of the conversion. All determinations were
carried out by triplicate and maximum error was
below 5%. Ratio of synthetic activity to hydrolytic
(
8
2
.3. Spectrophotometric assay of penicillin
acylase activity
Penicillin acylase activity was determined by measur-
ing the 6-APA released during the hydrolysis of penV
by the p-dimethyl-aminobenzaldehyde method
activity (S/H ratio) was calculated as V /Vh1.
S
(
Torres et al. 1999). In the case of the soluble
3
. Results and discussion
enzyme, enzyme activity was determined in a 300 ml
incubation mixture containing 1–2 mg of pure
enzyme and 58 mM penV in 50 mM potassium phos-
3.1. Effect of pH on kinetically controlled
synthesis of penicillin V
ꢁ
phate buffer pH 8.0 for 15 min at 40 C. The reaction
KCS of penV catalysed by SlPVA was carried out using
was stopped by the addition of 900 ml of 20% acetic
acid and immersion in an ice bath; the samples were
then centrifuged and 900 ml of the supernatant trans-
ferred to 300 ml of a solution of PDAB (0.5% in
methanol). Then the absorbance at 415 nm was
recorded for the determination of 6-APA concentra-
tion. All measurements were carried out by triplicate
and the maximum error was below 5%. One inter-
national activity unit (IU) was defined as the amount
of enzyme producing 1 mmol/min of 6-APA under the
assay conditions described above.
6
-APA as nucleophile and MPOA as activated acyl
donor. Since MPOA is slightly soluble in water, a small
amount of DMSO (2.7% v/v) as water-miscible organic
co-solvent was added to the reaction medium in order
to enhance its solubility. Time-course reaction profiles
were analyzed at different pH values ranging from 6
to 9 (Figure 1). In all cases, penV concentration
reached a maximum (Ymax), and then decreased grad-
ually due to hydrolysis as expected for a KCS of a
b-lactam antibiotic. Synthetic activity (V ) and hydro-
lytic activity (V ) were measured from each profile, as
S
h1
well as S/H ratio and Pmax (Table 1). As reported, S/H
ratio defines the nucleophile reactivity of 6-APA rela-
tive to water and determines the synthetic efficiency
of the acyl-transfer reaction (Youshko, Chilov, et al.
2
.4. Kinetically controlled synthesis of penicillin V
KCS of penV from 6-APA (as acyl acceptor) and MPOA
ꢁ
(as acyl donor) was carried out at 30 C in 100 mM
2
002). In this sense, S/H ratio was higher at pH 6.0
Tris/HCl buffer adjusted at different pH values.
Reactions were started by adding the enzyme
and 7.0 due to a reduction of hydrolytic activity at
neutral and acidic conditions, whereas S/H ratio was
abruptly reduced at basic pH values. These results
might be explained considering that catalytic bSer1 of
SlPVA (Torres-Bacete et al. 2015) should be deproto-
nated at alkaline conditions since pKa of a free
a-amine group ranges between 6.8 and 7.9 (Lee et al.
2000) and therefore formation of acyl-enzyme complex
would be favoured (Duggleby et al. 1995), and hydro-
lytic reactions (both MPOA ester hydrolysis as well as
penV hydrolysis) would be boosted. Similar results
were obtained in KCS of ampicillin catalyzed by
entrapped EcPGA on agar, where acyl-donor (phenyl-
glycine methyl ester, PGME) was barely hydrolysed at
pH 6.0, and then synthetic activity was promoted
(0.3–1.0 IU) to a 1.0 ml solution containing 20 mM of
MPOA and 6-APA at different concentrations ranging
from 40 to 200 mM. At predetermined time intervals, a
2
0 ll aliquot of the reaction mixture was withdrawn,
ꢁ
mixed with 20 ll of cold methanol (4 C), and further
centrifuged at 9000ꢂg for 5 min. The supernatant was
analyzed by HPLC (Agilent 1100) using a Luna C18 (2)
column 5 lm, 250 ꢂ 4.6 mm (Phenomenex, Torrance,
CA) with a 65% (v/v) methanol solution containing
0.05% (v/v) phosphoric acid as mobile phase. The flow
rate was fixed at 0.8 ml/min and the UV detector was
set at 260 nm. Retention times for the different sub-
strates and products were 3.8 min (6-APA), 6.4 min

4
D. HORMIGO ET AL.
Figure 1. Time-course of kinetically controlled synthesis of penicillin V (penV) catalysed by SlPVA at different pH values: (A) pH
6
.0; (B) pH 7.0; (C) pH 8.0; and (D) pH 9.0. PenV concentration is indicated in closed circles, whereas phenoxyacetic acid (POA)
ꢁ
concentration is indicated in opened circles. Reaction conditions: 30 C, 100 mM Tris/HCl buffer containing 2.7 % (v/v) DMSO,
100 mM 6-APA, 20 mM MPOA and 0.3 IU/ml SlPVA.
ꢀ
Table 1. Effect of pH on kinetically controlled synthesis of penV catalyzed by SlPVA .
Maximum yield (Ymax
)
pH
V
S
(mM/h)
Vh1 (mM/h)
S/H
(mM)
(%)
Reaction time for Ymax (h)
P
max (mM/h)
6
7
8
.0
.0
.0
.0
2.62
5.74
6.38
6.43
0.17
0.35
2.03
7.22
15.4
16.4
3.1
13.8
18.9
7.8
69.0
94.5
39.0
15.5
16.0
10.8
3.9
0.86
1.75
2.00
2.58
9
0.9
3.1
1.2
ꢀ
ꢁ
Reaction conditions: 30 C, 100 mM Tris/HCl buffer containing 100 mM 6-APA, 20 mM MPOA and 0.3 IU/ml SlPVA.
(
Ospina et al. 1996). In our case, maximum yield (Ymax
)
(Kasche 1986), and this fact must be taken into consid-
eration in our case. Since the side chain of penV lacks
an ionizable amino group in contrast to ampicillin and
cephalexin, the improved yield at pH 7.0 should be
likely attributed to an optimal enzyme conformation
of SlPVA that would allow a better binding of 6-APA
(nucleophile) and consequent exclusion of water that
would not compete with 6-APA for the acyl-enzyme
intermediate. Taking into account all these preliminary
results, further experiments were accomplished at a
neutral pH value.
was higher at pH 7.0 at a shorter reaction time (94.5%
at 10.8 h), compared to the result obtained at pH 6.0
(69% at 16 h), indicating that Pmax was two-fold lower
in acidic conditions (0.86 mM/h) than in neutral condi-
tions (1.75 mM/h). In this sense, reduced productivities
at low pH values have been also reported for KCS of
cephalexin and ampicillin catalyzed by different immo-
bilized EcPGA biocatalysts (Kim and Lee 1996b; Illanes
et al. 2003). Besides pH, enzyme and substrate proper-
ties can influence yield in a KCS of b-lactam antibiotics

BIOCATALYSIS AND BIOTRANSFORMATION
5
Figure 2. Time-course of kinetically controlled synthesis of penicillin V (penV) catalysed by SlPVA at different substrate molar
ratios: (A) 40 mM 6-APA/20 mM MPOA; (B) 75 mM 6-APA/20 mM MPOA; (C) 100 mM 6-APA/20 mM MPOA and (D) 200 mM 6-APA/
2
0 mM MPOA. PenV concentration is indicated in closed circles, whereas phenoxyacetic acid (POA) concentration is indicated in
ꢁ
opened circles. Reaction conditions: 30 C, 100 mM Tris/HCl buffer containing 2.7 % (v/v) DMSO and 0.3 IU/ml SlPVA.
ꢀ
Table 2. Effect of substrate molar ratio (6-APA to MPOA) on kinetically controlled synthesis of penV catalysed by SlPVA .
Maximum yield (Ymax
)
Molar ratio
2
V
S
(mM/h)
V
h1 (mM/h)
S/H
(mM)
(%)
Reaction time for Ymax (h)
P
max (mM/h)
5.29
5.84
5.74
5.24
ꢁ
0.55
0.42
0.35
0.32
9.6
13.9
16.4
16.4
13.1
15.9
18.9
18.4
65.5
79.5
94.5
92.0
7.8
9.0
10.8
10.9
1.67
1.77
1.75
1.69
3
5
.75
1
0
ꢀ
Reaction conditions: 30 C, 100 mM Tris/HCl pH 7.0 buffer containing 20 mM MPOA and 0.3 IU/ml SlPVA.6-APA concentration ranged from 40to 200 mM.
3
.2. Effect of substrate molar ratio on kinetically
were obtained at a molar ratio of 10. In such condi-
tions, the concentration of 6-APA would remain
almost constant along the process, allowing the S/H
to be maximum. In both cases, all MPOA was almost
used in the acyl-transfer reaction since yields in penV
synthesis were higher than 90%. On the contrary,
controlled synthesis of penicillin V
Time-course reaction profiles were also analyzed at dif-
ferent substrate molar ratios (6-APA to MPOA) ranging
from 2 to 10 at a fixed concentration of MPOA of
2
6
0 mM. As shown in Figure 2, the use of an excess of
-APA as nucleophile has a positive effect on anti-
when molar ratios were reduced to 3.75 and 2, Ymax
was diminished to 68% and 60%, respectively, mainly
biotic synthesis. In order to proof this, the dependence
of S/H ratio and Ymax on substrate molar ratio was
analyzed (Table 2). As observed, Ymax was higher at a
substrate molar ratio of 5, although similar results
due to a higher hydrolytic activity in such conditions.
In this sense, a high excess of 6-APA respect
to hydroxy-phenylglycine methyl ester (HPGM) as

6
D. HORMIGO ET AL.
Figure 3. Time-course of hydrolysis of MPOA (A) and kinetically controlled synthesis of penicillin V (B) catalysed by SlPVA in fully
ꢁ
aqueous reaction medium. Reaction conditions for MPOA hydrolysis: 30 C, 100 mM Tris/HCl pH 7.0 buffer, 20 mM MPOA, and
0
ꢁ
.2 IU/ml SlPVA. Reaction conditions for KCS of penicillin V: 30 C, 100 mM Tris/HCl pH 7.0 buffer, 200 mM 6-APA, 20 mM MPOA
and 0.2 IU/ml SlPVA.
acyl-donor has been reported to contribute to a spe-
cific enzyme inhibition towards ester hydrolysis in
amoxicillin synthesis catalysed by soluble EcPGA
3.3. Effect of enzyme concentration and organic
co-solvent on kinetically controlled synthesis of
penicillin V
(
Chow et al. 2005). A similar effect might happen in
the case of SlPVA, since 6-APA has been reported to
behave as non-competitive inhibitor in penV
First, the effect of enzyme concentration on the time-
course reaction profile was also analyzed (Figures 1(B)
and 4(D)) and the main KCS parameters were calcu-
lated (Table 3). As it might be expected, both syn-
thetic activity (V ) and hydrolytic activity (V ) were
a
hydrolysis (K ¼4 mM and K ¼5.8 mM) (Torres-Guzm ꢀa n,
is
ii
de la Mata, Arroyo, et al. 2001) and MPOA hydrolysis
could be inhibited as well. In the case of EcPGA, a
competitive inhibition of HPGM hydrolysis by 6-APA
S
h1
approximately 2-fold higher when enzyme concentra-
tion was increased 2-fold in the reaction. In addition,
Ymax was reached at a shorter reaction time (5.3 h),
but it was quite lower (64.5%) than the one obtained
with half amount of enzyme (94.5%). These results
may be related to the marked tendency of SlPVA to
self-associate in concentrated solutions to form aggre-
gates as previously reported (Hormigo et al. 2009). In
this situation, different SlPVA conformation in these
aggregates would lead to a less efficient binding of 6-
APA to the enzyme, thus favouring the access of water
to the acyl-enzyme intermediate and, consequently,
diminishing penV yield. Thus, an optimal enzyme con-
centration during KCS of penV is crucial to obtain
higher product yields and productivities. The detri-
mental effect of enzyme concentration on Ymax was
abolished when DMSO concentration was increased
from 2.7% to 40% (v/v) in the reaction medium
was established (K ¼7.23 mM) (Chow et al. 2005), sug-
is
gesting that the formation of the acyl-enzyme inter-
mediate would be blocked, and antibiotic synthesis
favoured (Gon c¸ alves, Sousa, et al. 2002). In our case,
inhibition of MPOA hydrolysis in the presence of a
high 6-APA concentration should not be discarded for
SlPVA, and that fact would explain the reduction of
hydrolysis rate at high substrate molar ratios. In fact,
inhibitory effect of 6-APA on enzyme-catalyzed
hydrolysis of MPOA was reported for EaPVA (Nam and
Ryu 1984). In order to support this hypothesis,
hydrolysis of MPOA catalyzed by SlPVA was carried
out in fully aqueous medium in absence of 6-APA. A
high hydrolytic rate of the ester (2.7 mM/h) and 49.6%
POA yield were observed at 7 h, whereas no MPOA
hydrolysis was detected in the KCS at the same reac-
tion conditions in the presence of 200 mM 6-APA
(
Figure 3). Likewise, similar V values were obtained in
S
the presence of DMSO at different concentrations
ranging from 2.7% to 20% (v/v) comparing with the
fully aqueous system, and no POA production was
observed at 2.5 h in each case (data not shown). As a
consequence of these results, further optimization of
KCS of penV was carried out with 100 mM 6-APA
nucleophile concentration, since best results of Ymax
and Pmax were obtained compared to 200 mM 6-APA
(
Figure 4). Although V was 3.5-fold lower with 40%
S
(v/v) DMSO, Ymax reached the same value (about
8
0–82%) independently of the enzyme concentration.
This result might be ascribed to the maintenance of a
low hydrolytic rate (V ¼0.14–0.19 mM/h) in reaction
h1
media containing a high co-solvent concentration.
This selective decrease of hydrolytic activity towards
(Table 2).

BIOCATALYSIS AND BIOTRANSFORMATION
7
Figure 4. Time-course of kinetically controlled synthesis of penicillin V (penV) catalysed by SlPVA at different enzyme
concentrations in presence of DMSO as co-solvent. (A) 0.3 IU/ml SlPVA, 40 % (v/v) DMSO; (B) 0.6IU/ml SlPVA, 40 % (v/v) DMSO;
(
C) 1.0 IU/ml SlPVA, 40 % (v/v) DMSO and (D) 0.6 IU/ml SlPVA, 2.7 % (v/v) DMSO. PenV concentration is indicated in closed circles,
ꢁ
whereas phenoxyacetic acid (POA) concentration is indicated in opened circles. Reaction conditions: 30 C, 100 mM Tris/HCl pH 7.0
buffer containing 100 mM 6-APA and 20 mM MPOA in the presence of DMSO as organic co-solvent.
ꢀ
Table 3. Effect of enzyme and DMSO concentrations on kinetically controlled synthesis of penV catalyzed by SlPVA .
Maximum yield (Ymax
)
DMSO (%) Enzyme concentration (IU/ml)
V
S
(mM/h)
V
h1 (mM/h)
S/H
(mM)
(%)
Reaction time for Ymax (h)
P
max (mM/h)
2
2
4
4
.7
.7
0
0
0
0.3
0.6
0.3
0.6
1.0
5.74
11.33
1.64
2.70
3.54
0.35
0.59
0.14
0.19
0.19
16.4
19.2
11.7
14.2
18.6
18.9
12.9
16.4
16.0
16.2
94.5
64.5
82.0
80.0
81.0
10.8
5.3
25.5
19.3
16.3
1.75
2.43
0.64
0.83
0.99
4
ꢀ
ꢁ
Reaction conditions: 30 C, 100 mM Tris/HCl pH 7.0 buffer containing 100 mM 6-APA and 20 mM MPOA.
both formed antibiotic and acyl-donor has been attrib-
uted in literature to the reduction of water activity
reduction of VS and increased S/H ratio in KCS of
cephalexin catalyzed by immobilized EcPGA in the
presence of 50% (v/v) DMSO (aw reduced to 0.683)
compared to fully aqueous medium (Aguirre et al.
2002). Besides, decreased productivities have been
usually reported by the presence of organic co-sol-
vents (Hyun et al. 1993; Kim and Lee 1996a; Illanes
and Fajardo 2001), as observed in our case (Table 3).
In general, all these variations on KCS parameters
should be mainly attributed to changes in enzyme
conformation produced by the co-solvent, and that
(aw) of the system after addition of a suitable co-solv-
ent (Aguirre et al. 2002; Illanes et al. 2002, 2003).
Although DMSO has been reported to reduce SlPVA
activity in penV hydrolysis at concentrations higher
than 20% (v/v) likely due to a denaturing effect
(Arroyo et al. 2000, 2002), reduction of synthetic and
hydrolytic activities in KCS of penV (Table 3) could be
related not only to protein denaturation but a reduc-
w
tion. Similarly, this effect could explain the strong

8
D. HORMIGO ET AL.
would depend not only on enzyme features (soluble
or immobilized) but on co-solvent properties.
Comparing our best results with other reports, Ymax of
penV with respect to the initial concentration of
MPOA was 94.5% (Figure 1(B)), higher than those
reported previously with SmPVA (85% yield based on
MPOA, using 200 mM 6-APA and 20 mM MPOA)
Funding
This work was supported by Grants BIO2008-03928, DEX-
580000-2008-31 from the Ministry of Education and Science
and Ministry of Science and Innovation of Spain, respectively,
and CTQ2014-60250-R project from Ministry of Economy,
Industry and Competitiveness of Spain and Grant S2009/PPQ-
1
752 from Comunidad Aut oꢀ noma de Madrid. This work also
supported by Consejer ꢀı a de Educaci oꢀ n, Juventud y Deporte,
Comunidad de Madrid; Ministerio de Ciencia e Innovaci oꢀ n;
Ministerio de Educaci oꢀ n y Ciencia and Secretar ꢀı a de Estado
de Investigaci oꢀ n, Desarrollo e Innovaci oꢀ n.
(
Koreishi et al. 2007) and EaPVA (12.2% yield based on
-APA, using 10 mM 6-APA and 80 mM MPOA) (Nam
and Ryu 1984).
6
4. Conclusions
ORCID
Although organic co-solvents are usually added to
improve product yields in KCS of b-lactam antibiotics,
presence of DMSO might be unnecessary in KCS of
penV catalysed by SlPVA using MPOA as acyl-donor,
and this could represent not only an economical
benefit but also an environmental advantage. As dem-
onstrated here, a fully aqueous system could be a bet-
ter option since a high 6-APA concentration is enough
to inhibit esterase activity of SlPVA towards MPOA and
therefore, DMSO would not be required to depress
this hydrolytic reaction. However, the addition of
DMSO up to 20% (v/v) concentration would allow an
enhanced solubility of the acyl-donor as observed for
MPOA, and the enzyme would not suffer any deacti-
vating effect in such conditions. That would open the
possibility of performing KCS of aliphatic penicillins
with hardly water-soluble methyl esters of the corre-
sponding fatty acids (such as methyl caprylate and
methyl caproate to be used in the KCS of penicillin K
and dihydroF, respectively), or even the KCS of other
N-acylated compounds employing different nucleo-
philes instead of 6-APA. In addition, an immobilized
form of SlPVA could be more suitable for this purpose
since it should show more stability and tolerate harsh
conditions such as the presence of denaturant co-sol-
vents, as well as high substrate and product concen-
trations (Garc ꢀı a-Gal ꢀa n et al. 2011). Moreover, an
immobilized enzyme could be recovered from the
reaction mixture, and then used repeatedly, leading to
a cost-effective process. In this sense, SlPVA has been
covalently linked to different epoxy-activated supports
Miguel Arroyo
http://orcid.org/0000-0002-1593-9817
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