Modulation of the microenvironment surrounding the active site of penicillin g acylase immobilized on acrylic carriers improves the enzymatic synthesis of cephalosporins

Article abstract of DOI:10.3390/molecules181114349

The catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled synthesis of β-lactam antibiotics are negatively affected upon immobilization on hydrophobic acrylic carriers. Two strategies have been here pursued to improve the synthetic performance of PGA immobilized on epoxy-activated acrylic carriers. First, an aldehyde-based spacer was inserted on the carrier surface by glutaraldehyde activation (immobilization yield = 50%). The resulting 3-fold higher synthesis/hydrolysis ratio (vs/vh1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit C and Sepabeads EC-EP, respectively) with respect to the unmodified support (vs/vh1 = 3.3 ± 0.4) was ascribed to a facilitated diffusion of substrates and products as a result of the increased distance between the enzyme and the carrier surface. A second series of catalysts was prepared by direct immobilization of PGA on epoxy-activated acrylic carriers (Eupergit C), followed by quenching of oxiranes not involved in the binding with the protein with different nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols). In most cases, this derivatization increased the synthesis/hydrolysis ratio with respect to the non derivatized carrier. Particularly, post-immobilization treatment with cysteine resulted in about 2.5-fold higher vs/vh1 compared to the untreated biocatalyst, although the immobilization yield decreased from 70% (untreated Eupergit C) to 20%. Glutaraldehydeand cysteine-treated Eupergit C catalyzed the synthesis of cefazolin in 88% (±0.9) and 87% (±1.6) conversion, respectively, whereas untreated Eupergit C afforded this antibiotic in 79% (±1.2) conversion.

Full text of DOI:10.3390/molecules181114349

Molecules 2013, 18, 14349-14365; doi:10.3390/molecules181114349
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Modulation of the Microenvironment Surrounding the Active
Site of Penicillin G Acylase Immobilized on Acrylic Carriers
Improves the Enzymatic Synthesis of Cephalosporins
Paolo Bonomi †, Teodora Bavaro, Immacolata Serra, Auro Tagliani, Marco Terreni * and
Daniela Ubiali *
Dipartimento di Scienze del Farmaco and Italian Biocatalysis Center, Università degli Studi di Pavia,
Via Taramelli 12, Pavia 27100, Italy; E-Mails: paolo.bonomi@utc.fr (P.B.);
teodora.bavaro@unipv.it (T.B.); immacolata.serra@unipv.it (I.S.); auro@ibiocat.eu (A.T.)
†
Present address: UTC—Université de Technologie de Compiègne, rue du Dr Schweitzer,
Compiègne 60200, France.
* Authors to whom correspondence should be addressed; E-Mails: marco.terreni@unipv.it (M.T.);
daniela.ubiali@unipv.it (D.U.); Tel.: +39-0382-987-265/987-889; Fax: +39-0382-422-975.
Received: 9 September 2013; in revised form: 20 October 2013 / Accepted: 28 October 2013 /
Published: 20 November 2013
Abstract: The catalytic properties of penicillin G acylase (PGA) from Escherichia coli in
kinetically controlled synthesis of β-lactam antibiotics are negatively affected upon
immobilization on hydrophobic acrylic carriers. Two strategies have been here pursued to
improve the synthetic performance of PGA immobilized on epoxy-activated acrylic
carriers. First, an aldehyde-based spacer was inserted on the carrier surface by
glutaraldehyde activation (immobilization yield = 50%). The resulting 3-fold higher
synthesis/hydrolysis ratio (vs/vh1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit® C and
Sepabeads® EC-EP, respectively) with respect to the unmodified support (vs/vh1 = 3.3 ± 0.4)
was ascribed to a facilitated diffusion of substrates and products as a result of the increased
distance between the enzyme and the carrier surface. A second series of catalysts was
prepared by direct immobilization of PGA on epoxy-activated acrylic carriers (Eupergit®
C), followed by quenching of oxiranes not involved in the binding with the protein with
different nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols). In
most cases, this derivatization increased the synthesis/hydrolysis ratio with respect to the
non derivatized carrier. Particularly, post-immobilization treatment with cysteine resulted
in about 2.5-fold higher vs/vh1 compared to the untreated biocatalyst, although the

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immobilization yield decreased from 70% (untreated Eupergit® C) to 20%. Glutaraldehyde-
and cysteine-treated Eupergit® C catalyzed the synthesis of cefazolin in 88% (±0.9) and
87% (±1.6) conversion, respectively, whereas untreated Eupergit® C afforded this
antibiotic in 79% (±1.2) conversion.
Keywords: penicillin G acylase; immobilization; microenvironment; epoxy acrylic
carriers; glutaraldehyde; hydrophilization
Abbreviations: 7-ACA, 7-amino-cephalosporanic acid; EDA, ethylendiamine; GA, glutaraldehyde;
PGK, penicillin G potassium salt; 7-ZACA, 7-amino-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-
carboxylic acid.
1. Introduction
Penicillin G acylase (PGA, E.C. 3.5.1.11) from E. coli is a N-terminal serine hydrolase mainly used
in the industrial production of 6-aminopenicillanic acid (6-APA) and 7-aminodesacetoxy-
cephaloporanic acid (7-ADCA) for the synthesis of β-lactam antibiotics [1].
Different strategies have been developed to enhance the properties of biocatalysts (e.g., stability),
including multimeric enzymes, to tailor them also for an industrial application [2,3]. Covalently
immobilized PGA on solid supports results in a more stable biocatalyst, which can be easily recovered
and reused [4–10]. However, immobilization generally exerts a detrimental effect on the synthetic
performance of PGA in the kinetically controlled acylation of β-lactam nuclei, particularly when epoxy
acrylic carriers such as Eupergit® C or Sepabeads® EC-EP are used. More hydrophilic carriers such as
glyoxyl agarose have been found superior because they generate a microenvironment more favorable
to the diffusion of the β-lactam nucleus into the active site [11,12].
Immobilization on glyoxyl agarose may be considered moderately complex at industrial scale [12]
since it is a multi-step procedure and it requires reagents and conditions which are plagued by difficult
handling and hazard issues, especially at a large scale (glycidol, sodium periodate, sodium
borohydride, alkaline pH). Conversely, for industrial purposes, commercial epoxy acrylic supports
appear to be almost ideal matrices since enzyme immobilization relies on a milder and more
straightforward procedure (covalent immobilization occurs directly between nucleophiles, mainly Lys
residues of the protein surface, and the epoxy-groups of the carrier) [13]. Therefore many efforts have
been addressed to improve the synthetic performances of epoxy acrylic carriers.
Differences between glyoxyl agarose and epoxy acrylic carriers become far less striking when
Eupergit® C or Sepabeads® EC-EP are functionalized with aldehyde groups, that is the same activation
commonly carried by the agarose support [11]. However, regardless of both the nature and the
activation of the support, PGA immobilization prevalently occurs through the enzyme surface bearing
the active site, although via different binding areas [11,14]. It is thus evident that other factors, besides
the binding chemistry and the nature of carrier, concur in determining the synthetic properties of
this biocatalyst.

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Steric hindrance promoted by the proximity of the support surface may prevent substrates from
accessing the catalytic site. In the case of PGA, β-lactam nuclei are bulky molecules that can hardly
reach the enzyme active site if this is not exposed to the medium. When covalent immobilization
occurs through a spacer between the enzyme and the support, steric problems can be frequently
circumvented [13,15]. Enzyme preparations where only one or a few residues of the protein are
involved in the enzyme-support covalent binding through the spacer (e.g., glutaraldehyde, dextran) do
not usually suffer from drastic loss of activity since the protein tends to behave as in its native state [15–17].
However, the lack of effect on enzyme activity [18] (due to a limited number of covalent bonds
between enzyme and support and thus to a negligible “rigidification” of the protein) frequently turns
into a quite poor stability of the immobilized enzyme that is thus not suitable for industrial
applications. Control of the experimental conditions and of the length and nature of the spacer are indeed
key points.
Alterations of the microenvironment in terms of charge or hydrophilicity can also exert a dramatic
effect on protein properties. Co-immobilization on glyoxyl agarose of PGA and polycationic polymers
have been performed to enhance the stability of the biocatalyst in organic solvents through the creation
of a sort of “hydrophilic shell” [19–24]. Modification of the final enzyme preparation with hydrophilic
compounds (typically amino acids) yields a fully inert support by preventing from any additional
interaction between the enzyme and the matrix [6,13].
The catalytic properties of enzymes are affected by immobilization; this technique can be a useful
tool to tune the enzyme performances [25,26]. When PGA is used in the kinetically controlled
synthesis of β-lactam antibiotics (or β-lactam intermediates), immobilization can influence the
synthetic performance. The synthetic efficiency of PGA is routinely assessed by determining the
synthesis/hydrolysis ratio (vs/vh1) of the acylation of a β-lactam nucleus with an ester or an amide
where s is the synthesis of the β-lactam antibiotic and h1 is the hydrolysis of the acyl donor (Scheme 1).
The highest transient yield achieved is negatively affected by h1 and by the hydrolysis of the final
product (h2) [27,28]. In particular, h2 is mainly involved at the end of the synthetic process, when the
concentration of the acylation product is predominant, while h1 is observable at the beginning of the
reaction when the concentration of the acyl donor is high (Scheme 1). Since immobilization of PGA
affects the vs/vh1 ratio, the evaluation of this value at the beginning of the reaction is routinely
considered a suitable descriptor of the biocatalyst efficiency, in agreement with previously reported
results [29–31].
We here described the preparation of two series of PGA-based catalysts on acrylic carriers and the
application of the most promising ones in the synthesis of cefazolin (Scheme 1). In the first set of
biocatalysts, a spacer was inserted on the carrier surface by glutaraldehyde activation to assess the
effect of an increased distance between the enzyme and the carrier surface on the catalytic properties
of PGA. The second group of biocatalysts was prepared by direct immobilization of PGA on
epoxy-activated acrylic carriers, followed by quenching of unreacted oxiranes with different
nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols) to assay whether a more
hydrophilic microenvironment correlates with higher synthetic performances.

Molecules 2013, 18
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Scheme 1. Kinetically controlled acylation of β-lactam nuclei (7-ACA and 7-ZACA,
respectively) catalyzed by PGA discussed throughout the text.
N
X
Ar= Ph
X=OH
Ar=
X=H
N
N
Ar
COOMe
N
(1-H-tetrazol-1-yl)-2-acetic acid
methyl ester
R-(-)-mandelic methyl ester
PGA
X
OH
H2O
h1
O
N
N
H2O
H
Ar
Enzyme
COOH
N
N
S
h1
O
COOH
7-ACA
s
7-ZACA
s
h2
h2
OH
HN
HN
S
N
O
S
N
CH3
S
O
N
N
N
OAc
O
O
N
COOH
COOH
N
N
Mandelyl-7-ACA
Cefazolin
Legend: s = synthesis; h1 = hydrolysis of the acyl donor [R-(−)-methyl mandelic ester or (1-H-tetrazol-1-yl)-
2-acetic methyl ester, respectively]; h2 = secondary hydrolysis.
2. Results and Discussion
2.1. Immobilization of PGA through a Spacer
Epoxy acrylic supports (Eupergit® C and Sepabeads® EC-EP) have been oxidized to their aldehyde
forms after conversion of the epoxy groups into diols via acid hydrolysis [11] (Scheme 2a–b). To
introduce a spacer between the enzyme and the surface of the carrier, aldehyde-Eupergit® C and
aldehyde-Sepabeads® EC-EP have been reacted with ethylendiamine and then with glutaraldehyde by
Schiff’s base chemistry (Scheme 2c) [32]. As a result, aldehyde groups of the amino-alkyl spacer
reacting with the amino groups of the protein are located far from the surface of the matrix.
Immobilization occurs, again, through the formation of Schiff’s bases generated from the reaction
between the enzyme amino groups and the aldehyde groups of the spacer. Chemical reduction of
imines to stable C-N bonds completes the two-step process (Scheme 2d–e) [8]. As a consequence of
the activation protocol, the resulting matrix has three different functionalities: the primary amino
groups, the fairly hydrophobic chain of glutaraldehyde and the aldehyde reactive groups. Depending
on the immobilization conditions, it has been reported that the first event of immobilization may have
different causes and involve different areas of the enzyme surface [33]. However, in all cases, this
immobilization protocol increases the distance between the carrier surface and the protein.
Scheme 2. Immobilization of PGA through a amino-alkyl spacer.
H2N
H
O
OH
OH
N
O
O
(a)
OH
(b)
(c)
N
H
N
H
O
O
H
O
OH
N
(d)
H
H
H
N
N
N
H
N
N
N
(e)
N
N
H
N
H
H
H
N
H
H
N
N
Experimental conditions: (a) 0.5 M H2SO4, r.t., 2 h, (see ref. [9]); (b) 100 mM NaIO4, r.t., 2 h, (see
ref. [9]); (c) reaction with ethylendiamine (EDA): 2 M EDA, pH 10, r.t., 2 h; NaBH4, 2 h. Reaction with
glutaraldehyde (GA): GA 25% v/v in H2O, pH 7, r.t., 16 h; (d) PGA (200 IU/g), pH 7, r.t., 3 h; (e) NaBH4,
pH 7, 30 min.

Molecules 2013, 18
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Our goal was to produce a biocatalyst with such a high degree of freedom to mimic the enzyme in
its native state, by reducing (or even overriding) any steric hindrance caused by the proximity of the
support surface to the active site. For this reason, in this case, immobilization was performed at
moderate ionic strength and neutral pH [12,33] to drive Schiff’s bases formation towards the terminal
amino groups of the enzyme, exclusively, without involving surface amino groups (mostly Lys). The
pH of the immobilization solution strongly affects the reactivity of the amino groups allowing for a
sort of “selection” of the amines involved in the reaction with the support. Being the pKa value of the
NH2 terminus around 7−8, at neutral pH this group is far more reactive than the average Lys residues
(whose pKa is estimated to be around 10). Therefore, this immobilization initially results in a single
amino group-support bond. Other bonds can then occur with Lys nearby the first imino bond to afford
a multipoint interaction, especially when highly activated carriers are used. It is plausible that the first
bond between PGA and the carrier occurs with the N-terminal-amino group of the α-chain (Glu1), as
the N-terminal-amino group of the β-chain (Ser1) is buried in the enzyme’s active site. A similar
scenario, where immobilization is designed to make the active site highly accessible to substrates, was
achieved by site directed mutagenesis introducing a flexible tag of three Lys alternating with three Gly
nearby the N-terminus [34,35].
It should not be underestimated that glutaraldehyde structure in aqueous solution is not limited to
the monomeric form but also dimers, trimers, polymers, cyclic hemiacetal can occur leading to many
different possible reaction mechanisms upon reaction of glutaraldehyde with proteins [36]. However,
each of these structures would be expected to form Schiff’s bases upon nucleophilic attack by Lys
residues in a protein that turns into an increased distance between the enzyme and the carrier [36].
Scheme 2 represents indeed a simplified sketch of immobilization of PGA through a glutaraldehyde-
based spacer.
Glyoxyl agarose was activated with ethylendiamine and glutaraldehyde according to the route
above described and then it was used for PGA immobilization as well. The synthetic efficiency of
PGA is routinely assessed by determining the synthesis/hydrolysis ratio (vs/vh1), as stated in the
Introduction (Scheme 1) [29–31,35]. This ratio depends on the affinity of the β-lactam nucleus for the
enzyme active centre and describes the percentage of synthesis at the beginning of the reaction
(percentage of acylating agent converted into the acylation product): a high value of vs/vh1 means that
the β-lactam nucleus is well adsorbed in the enzyme’s active site and the acyl moiety is efficiently
transferred on the β-lactam core. As the reference reaction for such an evaluation, the synthesis of
mandelyl-7-ACA (from 7-aminocephaloporanic acid, i.e., 7-ACA, and R-(−)-methyl mandelic ester)
was here considered (Scheme 1).
Results obtained in the synthesis of mandelyl-7-ACA catalyzed by PGA immobilized through the
amino-alkyl spacer on different carriers are depicted in Figure 1. For a comparative evaluation, both
vs/vh1 and percentage of conversion were reported for all the prepared PGA-based catalysts (with or
without the spacer) and the native enzyme.
From the inspection of Figure 1 it is evident that the presence of a spacer between the enzyme and
the support produced, in all cases, an enhancement of the synthetic catalytic properties of the
immobilized PGA in the studied reaction, as supported by the higher values of vs/vh1. Results
obtained with epoxy acrylic carriers upon functionalization with aldehyde groups and subsequent
ethylendiamine-glutaraldehyde activation is noteworthy: vs/vh1 of both acrylic carriers (9.7 ± 0.7 for

Molecules 2013, 18
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Eupergit® C and 10.9 ± 0.7 for Sepabeads®, respectively) reached the value of glyoxyl agarose (9.9 ± 1.0),
which is the highest performing carrier for PGA, since its synthetic behavior is comparable to that of
the native enzyme (9.6 ± 0.4). These data are consistent with a minor steric hindrance achieved
through the use of the spacer that also reduces the negative effect of the hydrophobic surface by
driving the catalytic site away from the carrier.
Figure 1. Selectivity of the PGA biocatalysts between the N-acylation reaction (vs) of
7-ACA and the hydrolysis of R-(−)-methyl mandelic ester (vh1).
Reaction conditions (saturating conditions): 4 °C, pH 6.5, acyl donor 10 mM, 7-ACA 50 mM.
Data reported above each column refer to the highest percentage of conversion. Percentage of
conversion was calculated as following: (vs/vs + vh1) × 100 (see Experimental). Immobilization:
loading = 200 IU/g (14.3 mg of protein). Legend: aldehyde activation (green) (from ref. [9]); epoxy activation
(red) (from ref. [9]); glutaraldeyde activation (purple).
Interestingly, if one considers the data of the set of PGA-base biocatalysts on Eupergit® C or
Sepabeads® EC-EP it clearly appears that when the acrylic supports carry the same activation of the
most favorable carrier (agarose) the vs/vh1 is higher than the value obtained with the epoxy activation.
The effect played by the nature of the support thus emerges as individual contribution to the enzyme
performance: the detrimental influence exerted by the hydrophobic matrix, although attenuated by
aldehyde functionalization, can be fully quenched only by the addition of a spacer. This evidence
confirms that the more exposed to the medium (and far from the hydrophobic support) is the catalytic
pocket, the higher is the synthetic performance of the biocatalyst.
It is worth mentioning that no difference was observed when PGA was immobilized on glyoxyl
agarose or on glutaraldehyde-activated agarose. In this case, the more hydrophilic nature of this carrier
has per sé a positive effect and the spacer does not further enhance the catalytic properties of PGA.
Data reported in Figure 1 were obtained when a large excess of β-lactam nucleus (50 mM) was used
in the PGA-catalyzed acylation (i.e., in “saturating” conditions) to enhance the achievement of the
highest conversion into the product. In this case the active site is “saturated” with the β-lactam nucleus
which can efficiently compete with water for the acyl-enzyme complex (Scheme 1). However,
even in “nonsaturating” conditions (β-lactam nucleus: 5 mM) glutaraldehyde-activated acrylic
carriers showed the highest vs/vh1 among all the considered immobilized biocatalysts (Figure S1,
Supplementary Materials).

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Immobilization yields on glutaraldehyde-activated carriers were moderate to high in all cases and
never below 50%.
2.2. Post-Immobilization Modification of Epoxy Acrylic Carriers (Eupergit® C)
From the results previously discussed it is evident that the catalytic behaviour of PGA depends on
several factors and, mostly important, that the interplay of immobilization techniques and experimental
conditions can modulate the synthetic performance of the enzyme when it is immobilized on acrylic
carriers till resembling the non immobilized enzyme. Again, the effect of the nature of the support has
been confirmed to play a pivotal role [11].
Being fairly hydrophobic, acrylic carriers negatively affect the catalytic properties of PGA in the
synthesis of β-lactam antibiotics. A more hydrophilic (and fully inert) support can be obtained after the
two-step immobilization (i.e., hydrophobic adsorption and covalent attachment) by blocking the
remaining epoxy groups of the carrier with hydrophilic nucleophiles (Scheme 3). This step can alter
the physical properties of the support surface, quenches the enzyme-support reaction and prevents
substrates from reacting with the support upon catalysis. Amino acids such as glycine are generally
used to this end [13]. We here used a representative series of different nucleophiles (amino acids,
amines, amino alcohols, thiols, amino thiols) to assay whether the hydrophilic/hydrophobic balance
correlates with the synthetic performances. To this aim, some reagents were selected to introduce a net
charge on the carrier (e.g., amino acids), others (e.g., amino alcohols) according to a polarity ranking,
whereas ethylendiamine (EDA), cysteamine and mercaptoethanol were useful to compare the effect of an
hydroxyl group against an amino group (for cysteamine and mercaptoethanol it is plausible that blocking
of the support epoxides occurs through the SH group, being a stronger nucleophile). Isobutylamine was the
negative control as this molecule concurs to increase the hydrophobic properties of the support. All
reactions were performed with Eupergit® C preparations at pH 9.5−10 to enhance the reactivity of the
nucleophiles. Epoxy groups are very stable under alkaline conditions for long-term incubations.
Scheme 3. Quenching of unreacted epoxy groups after immobilization.
H2N
H
H
O
OH
OH
N
N
H2N
H
O
H
epoxide blocking
OH
OH
N
N
O
O
OH
R2
R1
(a)
(b)
X
R
(Glycine)
(Aspartic acid)
(Lysine)
X=NH
X=NH
R=COOH
R=COOH
R1=R2=H
R1=CH2COOH
R2=H
X=NH
X=CH2S
X=NH
R=COOH
R=COOH
R=R1=H
R=OH
R1=(CH2)4NH2 R2=H
R1=NH2
R2=CH2OH
R1=R2=CH2OH
R2=H
(Cysteine)
(Ethanolamine)
(Tris)
X=NH
OH
X=NH
(Glucosamine)
R=R1=R2=
O
HO
HO
OH
(EDA)
X=NH
X=S
X=S
R=R1=H
R=R1=H
R=R1=H
R=H
R2=CH2NH2
R2=CH2NH2 (Cysteamine)
R2=CH2OH (Mercaptoethanol)
R2=CH2CH3 (Isobutylamine)
X=NH
R1=CH3
Experimental conditions: (a) 1–1.5 M phosphate buffer, pH 8, 24 h; (b) 1–3 M blocking reagent, 50 mM NaHCO3
pH 10 containing 50 mM phenylacetic acid, 24 h. Blocking reagents are given in brackets.

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According to established protocols, blocking reagents were added in very high concentration (1–3 M,
depending on their solubility) [6,13] at the end of the immobilization step (24 h) and kept under
stirring for further 24 h. For each blocking reagent, the residual activity of the biocatalyst after the
overall process (48 h) is depicted in Figure 2.
Figure 2. Residual activity and vs/vh1 of immobilized PGA on Eupergit® C after
post-immobilization quenching of unreacted epoxides.
Reaction conditions (nonsaturating conditions): 4 °C, pH 6.5, substrates (acyl donor and 7-ACA) 5 mM.
Legend: percentage of residual activity after post-immobilization blockage (red); vs/vh1 (blue).
Immobilization: loading = 200 IU/g (14.3 mg of protein).
In most cases the activity of the final enzyme preparation was sensitively decreased. The series of
amino acid blocking reagents appeared not to exert negative effect (glycine, aspartic acid and lysine)
with the exception of cysteine. In fact, after cysteine quenching, the immobilization yield was about
15% (30 IU/g).
Surprisingly, Tris was the outlier in a quite constant trend thus showing that in the series of amino
alcohols a correlation between the number of hydroxyl groups and the residual activity cannot
be found.
Loss of activity of the immobilized biocatalyst upon post-immobilization treatment can be
acceptable if it is compensated by a high synthetic performance. To verify this issue, all the enzyme
preparations were assayed in the synthesis of mandelyl-7-ACA (Scheme 1 and Figure 2). To better
highlight differences among all the tested biocatalysts, N-acylation of 7-ACA with R-(−)-methyl
mandelic ester was carried out in nonsaturating concentration (β-lactam nucleus: 5 mM). Under
saturating conditions (β-lactam nucleus: 50 mM), substrate diffusion to the catalytic site is maximized
and differences in the synthetic performances tend to collapse (see Figure S1, Supplementary Materials).
On the contrary, nonsaturating conditions highlight the accessibility of the active site and the
selectivity for the synthetic reaction (vs/vh1) can be better correlated, in this case, with the
post-immobilization treatment.

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Interestingly, if one compares the two series of data reported in Figure 2 it can be noticed that the
the less enzyme activity is retained after epoxide blocking, the higher is the vs/vh1. Amino acids did
not exert any remarkable effect on the residual activity of immobilized PGA and their selectivity for
the synthetic reaction was retained, being comparable to that of the unmodified biocatalyst (the only
exception is cysteine). All other blocking reagents resulted in an improvement, although to a different
extent, of the vs/vh1. The increased hydrophilicity of the microenvironment may be a plausible
explanation for the observed improvement (as supported by the trend of amino alcohols) but not
exhaustive, as suggested by the value found for isobutylamine.
The most relevant result was undoubtely provided by post-immobilization modification of the
biocatalyst with cysteine. In this case the vs/vh1 was by far higher (1.52) with respect to the untreated
biocatalyst (0.63) and very close to the value measured for the non immobilized PGA (1.87). However,
the very positive result of this biocatalyst in the synthesis was associated with a poor activity recovery
(about 15%) upon post-immobilization epoxide quenching (Figure 2).
In an attempt to find a balance between residual enzyme activity after cysteine blockage and
synthetic performance, we tested concentrations lower than 3 M (1.5 and 0.5 M) of this amino acid as
quenching reagent. Although with 0.5 M cysteine the enzymatic residual activity was increased by
2-fold (Figure 3), this result was not associated with the same improvement in the vs/vh1 (vs/vh1 = 5.5)
that was lower than the reference value (3 M cysteine).
Figure 3. Use of cysteine (3–0.5 M) as post-immobilization epoxide blocking.
Percentage of enzymatic activity recovery after blockage and vs/vh1 are reported (red and blue, respectively).
Reaction conditions (saturating conditions): 4 °C, pH 6.5, acyl donor 10 mM, 7-ACA 50 mM.
Immobilization: loading = 200 IU/g (14.3 mg of protein).
Overall, 1.5 M appeared to be the optimal value to conjugate enzyme activity recovery and
efficiency in the synthetic application (20% of recovered activity and vs/vh1 = 6.3).
Curves depicted in Figure 4 represent the theoretical yield (%) of mandelyl-7-ACA from the
acylation of 7-ACA upon increasing concentration of the β-lactam nucleus. This plot clearly shows
that different enzyme preparations can perform more similarly at the beginning of the reaction (when
the concentration of the β-nucleus is high), whereas as more as the reaction proceeds (and the β-lactam

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nucleus concentration decreases), the different ability of the examined catalysts in converting the β-lactam
nucleus into the acylation product is highlighted. This comparison can be very informative in the view
of preparative applications to address the choice of the biocatalyst. It is evident here that no difference
can be detected between PGA immobilized on Eupergit® C treated with 1.5 M cysteine and the non-
immobilized enzyme in saturating conditions. PGA immobilized on untreated Eupergit® C is much less
performing than the cysteine-treated counterpart both in nonsaturating and in saturating conditions.
Figure 4. Theoretical yield of R-(−)-mandelyl-7-ACA at increasing concentrations (5–50 mM) of 7-ACA.
Reaction conditions: 4 °C, pH 6.5, R-(−)-methyl mandelic ester: 5 mM. Theoretical yield was calculated as
following: (vs/vs + vh1) × 100 (see Experimental).
The difference between vs/vh1 of the PGA immobilized on Eupergit® C (3.3) and the cystein-treated
counterpart (6.3) (Figure 3) prompted us to a further investigation.
We hypothesized that the higher vs/vh1 of the cysteine-treated PGA preparation might be ascribed
to the higher substrate (β-lactam nucleus) concentration achieved in the active site as a result of a more
hydrophilic microenvironment. According to this, a drop of the rate of ester hydrolysis (vh1) and an
enhancement of the rate of synthesis (vs) could be expected since the β-lactam nucleus can more easily
“compete” with water for the acyl-enzyme complex (see Scheme 1).
To verify this, micromoles of product (mandelyl-7-ACA and mandelic acid) obtained by PGA-catalyzed
bioconversion in nonsaturating conditions were plotted against time course for the PGA immobilized
on Eupergit® C and the cystein-treated counterpart (1.5 M) (Figure 5).
Post-immobilization blocking with 1.5 M cysteine was also applied to Sepabeads® EC-EP and
Eupergit® C 250L. In both cases, vs/vh1 (in saturating conditions) were up to 2-fold higher than the
values achieved with the untreated biocatalysts and the percentage of conversion in the synthesis of
mandelyl-7-ACA was increased by about 10% (Figure 6). Sepabeads® EC-EP gave the same data
obtained with Eupergit®, whereas Eupergit® C 250L was slightly superior because of its larger pore [37]
dimensions which results in an enhancement of mass-transfer.

Molecules 2013, 18
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Figure 5. Vs (square) and vh1 (triangle) of the reaction catalyzed by PGA immobilized on
Eupergit® C before (red) and after quenching with 1.5 M cysteine (green).
Eupergit Cys (R-mandelic acid )Vh1=0.21
Eupergit Cys (mandelyl-7-ACA) Vs=0.26
Eupergit C (R-mandelic acid) Vh1=1.15
Eupergit C (mandelyl-7-ACA) Vs=0.5
18
16
14
12
10
8
6
4
2
0
0
2
4
6
8
10
12
14
Time (min)
Reaction conditions (non-saturating conditions): 4 °C, pH 6.5, substrates (acyl donor and 7-ACA) 5 mM;
immobilized PGA = 0.15 g.
Figure 6. Vs/vh1 and percentage of conversion (synthesis of mandelyl-7-ACA) of PGA
immobilized on epoxy acrylic carriers (Eupergit® C, Sepabeads® EC-EP and Eupergit®
250L) before and after quenching with cysteine.
Reaction conditions (saturating conditions): 4 °C, pH 6.5, acyl donor 10 mM, 7-ACA 50 mM. Data reported
above each column refer to the highest percentage of conversion. Percentage of conversion was calculated as
following: (vs/vs + vh1) × 100 (see Experimental). Legend: epoxy activation (red) (from ref. [9]); 1.5 M
cysteine quenching (green).
2.3. Synthesis of Cefazolin
PGA immobilized on Eupergit® C blocked with 1.5 M cysteine was used in the synthesis of
Cefazolin at a preparative concentration (Scheme 1). N-acylation was performed with a 3:1 excess of
the ester to achieve the complete conversion of the β-lactam nucleus (7-ZACA, 50 mM) into the product. In
this synthesis the acylation product is not hydrolyzed by the PGA from E. coli [11,29,30] and thus its
concentration remains almost constant after reaching the highest value. For this reason, h2 is negligible
(see Scheme 1), the percentage of conversion is only influenced by the hydrolysis of the acylating ester

Molecules 2013, 18
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(vh1) and the yield depends exclusively on the vs/vh1 ratio and therefore on the catalytic behavior of
the biocatalyst.
Table 1 reports the comparison of four enzyme preparations: PGA immobilized on Eupergit® C as
such and upon glutaraldehyde- and cysteine-activation; PGA immobilized on glyoxyl agarose was also
considered as it represents the highest performing PGA-based catalyst.
Table 1. Percentage of conversion obtained in the synthesis of Cefazolin catalyzed by
different PGA preparations (see also Scheme 1).
Support
Eupergit® C
Eupergit® C
Eupergit® C
Agarose
Activation
Epoxy a
Glutaraldehyde
“Epoxy-Cysteine”
Aldehyde a
Time (min)
180
Conversion (SD%)
79% (1.2)
330
270
330
88% (0.9)
87% (1.6)
90% (1.8)
Experimental conditions: 150 mM acyl donor, 50 mM β-lactam nucleus (7-ZACA), PGA: 50 IU. The
reaction was initiated at pH 7.5 to ensure the complete solubilization of 7-ZACA and then maintained at 6.5
(see ref. [11]). a From ref. [11].
3. Experimental
3.1. General
The extracts of PGA from E. coli as well as penicillin G potassium salt (PGK), the β-lactam nuclei,
the analytical standards of cefazolin and (1-H-tetrazol-1-yl)-2-acetic acid methyl ester were kindly
provided by ACS Dobfar SpA (Tribiano, Milano, Italy). The protein concentration of PGA extract was
100 mg/mL with a specific activity towards PGK of 14 IU/mg. Cross-linked 6% agarose beads
(Sepharose 6B-CL) was from Amersham Biosciences AB (Uppsala, Sweden). Sepabeads EC-EP was a
gift of Resindion (Binasco, Milano, Italy), while Eupergit® C was from Röhm Pharma (Darmstadt,
Germany). R-(−)-Methyl mandelic ester, and all other reagents and solvents (HPLC grade) were
purchased from Sigma-Aldrich (Milano, Italy).
The pH of the solutions during the enzymatic hydrolyses and syntheses was kept constant by using
an automatic titrator 718 Stat Titrator from Metrohm (Herisau, Switzerland). HPLC analyses were run
on a BIO-TEK Kontron (Milano, Italy) instrument equipped with a UV detector 535. The column was
a RP18 Interchrom 300 × 3.9 mM (5 mM). Protein concentration was determined by the Bradford assay
using bovine serum albumin as standard on a Shimadzu spectrophotometer UV 1601 (Milano, Italy).
3.2. Enzymatic Activity Assay
The hydrolytic activity of PGA was measured in international units (IU). One IU corresponds with
the amount of enzyme that liberates one mmol of phenylacetic acid per minute from PGK, as
previously reported [34].
3.3. Enzyme Immobilization
All enzyme derivatives were prepared by loading 14.3 mg of enzyme per gram of carrier
(corresponding to about 200 IU).

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3.3.1. Immobilization of PGA on Epoxy Acrylic Supports
The epoxy-activated acrylic resins (Eupergit® C and Sepabeads® EC-EP) were used according to
the supplier indications. Immobilization was carried out at high phosphate concentration (1–1.5 M),
pH 8.0, for 24 h at room temperature as previously described [11].
3.3.2. Immobilization of PGA on Aldehyde Supports
Preparation of aldehyde agarose and immobilization were performed according to the procedure
previously described [8]. Similarly, starting from epoxy acrylic carriers, aldehyde activation and
immobilization were done according to the procedure previously reported [11].
3.3.3. Immobilization of PGA on Glutaraldehyde-Activated Acrylic Resins
Immobilization of PGA on supports activated with glutaraldehyde was performed according to the
procedure previously described, starting from aldehyde carriers [32]. Activation and immobilization
was performed in three steps:
Step 1. Reaction of aldehyde activated carriers with ethylenediamine. The aldehyde support (3 g), was
suspended in 2 M ethylenediamine pH 10.0 (38.7 mL) and kept at room temperature under mechanical
stirring for 2 h. Then, NaBH4 (43 mg, 1 mg per mL of suspension) was added and the reaction mixture
was maintained under stirring for additional 2 h. The support was then filtered under reduced pressure
on a glass filter, washed with distilled water and used for the activation with glutaraldehyde.
Step 2. Activation with glutaraldehyde. The aminated carrier (3 g) was suspended in 0.2 M
phosphate buffer pH 7.0 (3.4 mL), then a solution of 25% (v/v) glutaraldehyde (5.1 mL) was added.
The mixture was kept under mechanical stirring for 16 h at room temperature in absence of light. The
resin was filtered, washed with distilled water and immediately used for enzyme immobilization.
Step 3. Immobilization of PGA on carriers activated with glutaraldehyde. The glutaraldehyde
activated carrier (4 g) was suspended in 50 mM bicarbonate buffer pH 7.0 (22.8 mL) containing 100 mM
phenylacetic acid. The appropriate amount of enzyme extract to have 200 IU/g was added and
maintained under stirring for 3 h at room temperature. The suspension was diluted with a solution of
NaBH4 in 50 mM bicarbonate buffer (28.5 mL, 2 mg/mL) containing 100 mM phenylacetic acid, pH
was adjusted to 10.0 by adding NaOH. After 30 minutes, the enzyme was filtered, washed with 10 mM
phosphate buffer pH 5.0 and subsequently with distilled water.
3.4. Modification of Epoxy-Activated Acrylic Carriers
The immobilized enzyme (5 g) was suspended in a solution (28.6 mL) of the suitable amount of
blocking reagent dissolved in 50 mM bicarbonate buffer containing 50 mM phenylacetic acid (28.6 mL),
the pH was adjusted to 10.0 with NaOH and the suspension was kept under gentle stirring for 24 h.
The enzyme preparation was filtered, washed with 100 mM phosphate buffer pH 7.0 and then with
distilled water.

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3.5. Enzymatic Reactions
(a) The vs/vh1 ratio of the N-acylation of 7-ACA with R-(−)-methyl mandelic ester and the
percentage of synthesis were evaluated by measuring the initial rate of the synthesis (vs) of the
acylation product and of the rate of ester hydrolysis (vh1) to give R-mandelic acid at the beginning of
the reaction (before 20% of ester was converted), according to the general procedure previously
reported [34]. The reactions were monitored by HPLC analysis at 220 nm (eluent: 20% CH3CN in
10 mM phosphate buffer pH 3.2). The percentage of synthesis was calculated using the following
equation: [vs/(vs + vh1)] × 100.
(b) The synthesis of cefazolin was performed by N-acylation of 7-ZACA with (1-H-tetrazol-1-yl)-2-acetic
acid methyl ester, according to the procedure previously reported [11,13]. The reactions were
monitored by HPLC analysis at 274 nm (eluent: 20% CH3CN in 10 mM phosphate buffer pH 3.2).
3.6. Stability of the Immobilized Preparations
Each immobilized enzyme preparation (500 mg) was added to a solution (5 mL) containing 10 mM
phosphate buffer and 40% of MeOH (v/v) at pH 7.5 or 10 mM phosphate buffer at pH 7.5. The
mixture was stirred at room temperature or at 50 °C, respectively, for 48 h. At fixed times, 500 µL of
suspension were withdrawn and the residual activity was evaluated by standard activity assay (PGK test).
4. Conclusions
Acrylic carriers bearing epoxy groups such as Eupergit® C and Sepabeads® EC-EP rely on a
straightforward procedure for enzyme immobilization. In the case of PGA, however, immobilization
on those carriers negatively affects the synthetic properties of the enzyme. The introduction of a spacer
between the enzyme and the support resulted in an improvement of the synthetic performance of the
biocatalyst, consistently with the increased distance between the enzyme and the carrier surface. This
result was in agreement with that obtained when a specific amino acid tag was introduced on the
protein surface via site directed mutagenesis to generate a preferential immobilization point. However,
immobilization through glutaraldehyde is a complex multi-step procedure which is hardly feasible for
applicative purposes.
Immobilization on acrylic epoxy carriers, followed by post immobilization treatment with the
appropriate quenching reagent, is far easier to perform also at a large scale. Post-immobilization
quenching of oxiranes by reaction with hydrophilic nucleophiles generally increased the selectivity of
the enzyme for the synthesis. This result was (partly) ascribed to the generation of a more hydrophilic
microenvironment which can “compensate” for the detrimental effects exerted by the hydrophobic
support. However, a drop in the enzyme recovered activity was registered in some cases. Blocking of
the epoxy acrylic carriers (Eupergit® C, Sepabeads® EC-EP and Eupergit® C 250L) with cysteine
resulted in high performing biocatalysts, even comparable with the non-immobilized enzyme but with
the advantage of a heterogeneous catalysis.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/11/14349/s1.

Molecules 2013, 18
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Acknowledgments
We thank ACS Dobfar S.p.A (Tribiano, Milano, Italy) and Resindion S.r.l. (Binasco, Milano, Italy)
for their generous gift of PGA and Sepabeads® EC-EP, respectively.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Chandel, A.K.; Rao, L.V.; Narasu, M.L.; Singh, O.V. The realm of penicillin G acylase in
β-lactam antibiotics. Enzyme Microb. Technol. 2008, 42, 199–207.
2. Fernandez-Lafuente, R. Stabilization of multimeric enzymes: Strategies to prevent subunit
dissociation. Enzyme Microb. Technol. 2009, 45, 405–418.
3. Iyer, P.V. Ananthanarayan, L. Enzyme stability and stabilization-Aqueous and non-aqueous
environment. Process Biochem. 2008, 43, 1019–1032.
4. Kallenberg, A.I.; Van Rantwijk, F.; Sheldon, R.A. Immobilization of penicillin G acylase: the key
to optimum performance. Adv. Synth. Catal. 2005, 347, 905–926.
5. Guisàn, J.M.; Alvaro, G.; Fernàndez-Lafuente, R.; Rosell, C.M.; Garcia, J.L.; Tagliani, A.
Stabilization of heterodimeric enzyme by multipoint covalent immobilization: Penicillin G
acylase from Kluyvera citrophila. Biotechnol. Bioeng. 1993, 42, 455–464.
6. Mateo, C.; Abiàn, O.; Fernàndez-Lorente, G.; Pedroche, J.; Fernàndez-Lafuente, R.; Guisàn, J.M.;
Tam, A.; Daminati, M. Epoxy Sepabeads: A novel epoxy support for stabilization of industrial
enzymes via very intense multipoint covalent attachment. Biotechnol. Prog. 2002, 18, 629–634.
7. Mateo, C.; Fernàndez-Lorente, G.; Abiàn, O.; Fernàndez-Lafuente, R.; Guisàn, J.M.
Multifunctional epoxy supports: a new tool to improve the covalent immobilization of proteins.
The promotion of physical adsorptions of proteins on the supports before their covalent linkage.
Biomacromolecules 2000, 1, 739–745.
8. Guisàn, J.M. Aldehyde-agarose gels as activated supports for immobilization-stabilization of
enzymes. Enzyme Microb. Technol. 1988, 10, 375–382.
9. Alvaro, G.; Fernàndez-Lafuente, R.; Blanco, R.M.; Guisàn, J.M. Immobilization-stabilization of
penicillin G acylase from Escherichia coli. Appl. Biochem. Biotechnol. 1990, 26, 181–195.
10. Mateo, C.; Abiàn, O.; Bernedo, M.; Cuenca, E.; Fuentes, M.; Fernàndez-Lorente, G.;
Palomo, J.M.; Grazù, V.; Pessela, B.C.C.; Giacomini, C.; et al. Some special features of glyoxyl
supports to immobilize proteins. Enzyme Microb. Technol. 2005, 37, 456–462.
11. Temporini, C.; Bonomi, P.; Serra, I.; Tagliani, A.; Bavaro, T.; Ubiali, D.; Massolini, G.;
Terreni, M. Characterization and study of the orientation of immobilized enzymes by tryptic
digestion and HPLC-MS: design of an efficient catalyst for the synthesis of cephalosporins.
Biomacromolecules 2010, 11, 1623–1632.
12. Mateo, C.; Palomo, J.M.; Fuentes, M.; Betancor, L.; Grazù, V.; Lòpez-Gallego, F.;
Pessela, B.C.C.; Hidalgo, A.; Fernàndez-Lorente, G.; Fernàndez-Lafuente, R.; et al. Glyoxyl
agarose: a fully inert and hydrophilic support for immobilization and high stabilization of
proteins. Enzyme Microb. Technol. 2006, 39, 274–280.

Molecules 2013, 18
14364
13. Mateo, C.; Grazù, V.; Palomo, J.M.; Lopez-Gallego, F.; Fernàndez-Lafuente, R.; Guisàn, J.M.
Immobilization of enzymes on heterofunctional epoxy supports. Nat. Protoc. 2007, 2, 1022–1033.
14. Serra, I.; Ubiali, D.; Cecchini, D.A.; Calleri, E.; Albertini, A.M.; Terreni, M.; Temporini, C.
Assessment of immobilized PGA orientation via the LC-MS analysis of tryptic digests of the wild
type and its 3K-PGA mutant assists in the rational design of a high-performance biocatalyst.
Anal. Bioanal. Chem. 2013, 405, 745–753.
15. Penzol, G.; Armisen, P.; Fernàndez-Lafuente, R.; Rodes, L.; Guisàn, J.M. Use of dextrans as long
and hydrophilic spacer arms to improve the performance of immobilized proteins acting on
macromolecules. Biotechnol. Bioeng. 1998, 60, 518–523.
16. Fernàndez-Lafuente, R.; Rosell, C.M.; Rodriguez, V.; Guisàn, J. M. Strategies for enzyme
stabilization by intramolecular crosslinking with bifunctional reagents. Enzyme Microb. Technol.
1995, 17, 517–523.
17. Adriano, W.S.; Filho, E.H.C.; Silva, J.A.; Giordano, R.L.C.; Goncalves, L.R.B. Stabilization of
penicillin G acylase by immobilization on glutaraldehyde-activated chitosan. Brazil. J. Chem. Eng.
2005, 22, 529–538.
18. Fernàndez-Lorente, G.; Terreni, M.; Mateo, C.; Bastida, A.; Fernàndez-Lafuente, R.;
Dalmases, P.; Huguet, J.; Guisàn, J.M. Modulation of lipase properties in macro-aqueous systems
by controlled enzyme immobilization: Enantioselective hydrolysis of a chiral ester by
immobilized Pseudomonas lipase. Enzyme Microb. Technol. 2001, 28, 389–396.
19. Fernàndez-Lafuente, R.; Rosell, C.M.; Caanan-Haden, L.; Rodes, L.; Guisàn, J.M.
Facile synthesis of artificial enzyme nano-environments via solid-phase chemistry of immobilized
derivatives: dramatic stabilization of penicillin acylase versus organic solvents. Enzyme Microb.
Technol. 1999, 24, 96–103.
20. Jin, X.; Wu, Q.; Chen, Q.; Chen, C.X.; Lin, X.F. Immobilization of penicillin G acylase on a
composite carrier with a biocompatible microenvironment of chitosan. J. Chem. Technol.
Biotechnol. 2008, 83, 1710–1716.
21. Montes, T.; Grazù, V.; Manso, I.; Galan, B.; Lòpez-Gallego, F.; Gonzàlez, R.; Hermoso, J.A.;
Garcia, J.L.; Guisàn, J.M.; Fernàndez-Lafuente, R. Improved stabilization of genetically modified
penicillin G acylase in the presence of organic cosolvents by co-immobilization of the enzyme
with polyethyleneimine. Adv. Synth. Catal. 2007, 349, 459–464.
22. Zhou, C.; Zhu, S.; Wu, X.; Jiang, B.; Cen, T.; Shen, S. Post-immobilization of modified
macromolecular reagents using assembled penicillin acylase for microenvironmental regulation of
nanopores and enhancement of enzyme stability. Biotechnol. Bioproc. Eng. 2010, 15, 376–382.
23. Fuentes, M.; Pessela, B.C.C.; Maquiese, J.V.; Ortiz, C.; Segura, R.L.; Palomo, J.M.; Abiàn, O.;
Torres, R.; Mateo, C.; Fernàndez-Lafuente, R.; et al. Reversible and strong immobilization of proteins
by ionic exchange on supports coated with sulfate-dextran. Biotechnol. Prog. 2004, 20, 1134–1139.
24. Wilson, L.; Illanes, A.; Abiàn, O.; Pessela, B.C.C.; Fernàndez-Lafuente, R.; Guisàn, J.M.
Co-aggregation of penicillin G acylase and polyionic polymers: An easy methodology to prepare
enzyme biocatalysts stable in organic media. Biomacromolecules 2004, 5, 852–857.
25. Rodrigues, R.C.; Ortiz, C.; Berenguer-Murcia, A.; Torres, R.; Fernández-Lafuente, R. Modifying enzyme
activity and selectivity by immobilization. Chem. Soc. Rev. 2013, 42, 6290–6307.

Molecules 2013, 18
14365
26. Rodrigues, R.C.; Berenguer-Murcia, A.; Fernandez-Lafuente, R. Coupling chemical modification
and immobilization to improve the catalytic performance of enzymes. Adv. Synth. Catal. 2011,
353, 2216–2238.
27. Kasche, V. Mechanism and yields in enzyme catalysed equilibrium and kinetically controlled
synthesis of β-lactam antibiotics, peptides and other condensation products. Enzyme Microb.
Technol. 1986, 8, 4–16.
28. Kasche, V.; Haufler, U.; Riechmann, L. Equilibrium and kinetically controlled synthesis with
enzymes: Semisynthesis of penicillins and peptides. Meth. Enzymol. 1987, 136, 280–292.
29. Estruch, I.; Tagliani, A.R.; Guisàn, J.M.; Fernàndez-Lafuente, R.; Alcàntara, A.R.; Toma, L.;
Terreni, M. Immobilization of the acylase from Escherichia coli on glyoxyl-agarose gives
efficient catalyst for the synthesis of cephalosporins. Enzyme Microb. Technol. 2008, 42, 121–129.
30. Terreni, M.; Ubiali, D.; Bavaro, T.; Pregnolato, M.; Fernàndez-Lafuente, R.; Guisàn, J.M.
Enzymatic synthesis of cephalosporins. The immobilized acylase from Arthrobacter viscosus: A
new useful biocatalyst. Appl. Microbiol. Biotechnol. 2007, 77, 579–587.
31. Terreni, M.; Tchamkam, J.G.; Sarnataro, U.; Rocchietti, S.; Fernàndez-Lafuente, R.; Guisàn, J.M.
Influence of substrate structure on PGA-catalyzed acylations. Evaluation of different approaches
for the enzymatic synthesis of cefonicid. Adv. Synth. Catal. 2005, 347, 121–128.
32. Guisàn J.M.; Penzol G.; Armise P.; Bastida A.; Blanco R.M.; Fernàndez-Lafuente R.;
Garcia-Giunceda E. Immobilization of Enzymes and Cells; The Humana Press Inc: New York,
NY, USA, 1997; pp. 261–275.
33. Barbosa O.; Torres R.; Ortiz C.; Berenguer-Murcia A.; Rodrigues R.C.; Fernandez-Lafuente R.
Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to
opportunities in tuning enzyme properties. Biomacromolecules 2013, 14, 2433–2462.
34. Serra, I.; Cecchini, D.A.; UbiaIi, D.; Manazza, E.M.; Albertini, A.M.; Terreni, M. Coupling of
site-directed mutagenesis and immobilization for the rational design of more efficient biocatalysts:
The case of immobilized 3G3K PGA from E. coli. Eur. J. Org. Chem. 2009, 1384–1389.
35. Scaramozzino, F.; Estruch, I.; Rossolillo, P.; Terreni, M.; Albertini, A.M. Improvement of
catalytic properties of Escherichia coli penicillin G acylase immobilized on glyoxyl agarose by
addition of a six-amino-acid tag. Appl. Environ. Microbiol. 2005, 71, 8937–8940.
36. Migneault, I.; Dartiguenave, C.; Bertrand, M.J.; Waldron, K.C. Glutaraldehyde: behaviour in
aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques
2004, 37, 790–802.
37. Boller, T.; Meier, C.; Menzler, S. EUPERGIT oxirane acrylic beads: How to make enzymes fit for
biocatalysis. Org. Process Res. Dev. 2002, 6, 509–519.
Sample Availability: Samples of immobilized PGA on glyoxyl agarose and on epoxy acrylic carriers
are available from the Authors.
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