Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin

Article abstract of DOI:10.1016/j.molcatb.2010.06.014

α-Amino ester hydrolases (AEH) are a small class of proteins, which are highly specific for hydrolysis or synthesis of α-amino containing amides and esters including β-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin. A BLAST search revealed the sequence of a putative glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase 93% identical to a known AEH from Xanthomonas citri. The gene, termed gaa, was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913 and the corresponding protein was expressed into Escherichia coli. The purified protein was able to perform both hydrolysis and synthesis of a variety of α-amino β-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 °C and pH 6.8, with kinetic parameters of k_cat of 72.5 s^-1 and K_M of 1.1 mM. The synthesis parameters α, β_o, and γ for ampicillin, determined here first for this class of proteins, are α = 0.25, β_o = 42.8 M^-1, and γ = 0.23, and demonstrate the excellent synthetic potential of these enzymes. An extensive study of site-directed mutations around the binding pocket of X. campestris pv. campestris AEH strongly suggests that mutation of almost any first-shell amino acid residues around the active site leads to inactive enzyme, including Y82, Y175, D207, D208, W209, Y222, and E309, in addition to those residues forming the catalytic triad, S174, H340, and D307.

Full text of DOI:10.1016/j.molcatb.2010.06.014

Journal of Molecular Catalysis B: Enzymatic 67 (2010) 21–28
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Amino ester hydrolase from Xanthomonas campestris pv. campestris,
ATCC 33913 for enzymatic synthesis of ampicillin
Janna K. Blum, Andreas S. Bommarius∗
School of Chemical and Biomolecular Engineering, Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology,
315 Ferst Drive, Atlanta, GA 30332-0363, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
˛-Amino ester hydrolases (AEH) are a small class of proteins, which are highly specific for hydrol-
ysis or synthesis of ˛-amino containing amides and esters including ˇ-lactam antibiotics such as
ampicillin, amoxicillin, and cephalexin. A BLAST search revealed the sequence of a putative glutaryl
7-aminocephalosporanic acid (GL-7-ACA) acylase 93% identical to a known AEH from Xanthomonas citri.
The gene, termed gaa, was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp.
strain ATCC 33913 and the corresponding protein was expressed into Escherichia coli. The purified protein
was able to perform both hydrolysis and synthesis of a variety of ˛-amino ˇ-lactam antibiotics including
(R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 ◦C and pH 6.8, with kinetic
parameters of kcat of 72.5 s−1 and KM of 1.1 mM. The synthesis parameters ˛, ˇo, and ꢀ for ampicillin, deter-
mined here first for this class of proteins, are ˛ = 0.25, ˇo = 42.8 M−1, and ꢀ = 0.23, and demonstrate the
excellent synthetic potential of these enzymes. An extensive study of site-directed mutations around the
binding pocket of X. campestris pv. campestris AEH strongly suggests that mutation of almost any first-
shell amino acid residues around the active site leads to inactive enzyme, including Y82, Y175, D207,
D208, W209, Y222, and E309, in addition to those residues forming the catalytic triad, S174, H340, and
D307.
Received 14 March 2010
Received in revised form 28 June 2010
Accepted 30 June 2010
Available online 6 July 2010
Keywords:
Amino ester hydrolase
ˇ-Lactam antibiotics
Ampicillin
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
˛-Amino ester hydrolase (AEH) catalyzes the synthesis and
hydrolysis of esters and amides of ˛-amino acids, and can be used
Semi-synthetic ˇ-lactam antibiotics (mostly penicillins and
cephalosporins) make up approximately 65% of the total world
market of $15 billion for antibiotics [1]. One of the most effi-
cient ways to produce semi-synthetic ˇ-lactam antibiotics is
via biocatalytic acylation of a ˇ-lactam moiety, for example 6-
aminopenicillanic acid (6-APA), with an activated carboxylic acid,
for example (R)-phenylglycine methyl ester ((R)-PGME), as shown
in Scheme 1. DSM Anti-Infectives BV (Delft, Netherlands) currently
manufactures amoxicillin, cephalexin, and cefadroxil with an enzy-
matic process that utilizes penicillin G acylase (PGA, EC 3.5.1.11) [2].
Improved enzymatic production processes lead to lower cost and
more environmentally benign production of semi-synthetic antibi-
otics when compared to traditional chemical synthesis [1,3–10] and
the existing enzymatic synthesis.
as an alternative to the industrially employed penicillin G acylase
for the synthesis and hydrolysis of ˛-amino containing antibiotics.
While already discovered in 1974 by Takahashi et al. [11], AEHs
from both Acetobacter turbidans (ATCC 9325) and Xanthomonas
citri (IFO 3835) have only recently been cloned, overexpressed
in Escherichia coli, crystallized [5,9,10], and characterized as to
substrate specificity. These enzymes are serine hydrolases with
a classical Ser-His-Asp catalytic triad and belong to the struc-
tural type of ˛/ˇ-hydrolases [6,9,10]. The AEHs are unique in their
specificity toward ˛-amino groups; this specificity has been asso-
ciated with an acidic carboxylate cluster (D208, E309, D310) in the
enzyme’s active site. The current understanding of the carboxylate
cluster focuses on its involvement in the recognition of the ˛-amino
group on the substrate, thus positioning the substrate for catalysis
[5]. Since only two AEHs have been fully characterized so far, it is of
interest to further expand the number of characterized species in
this class to enable combinatorial or data-driven protein engineer-
ing techniques such as gene recombination [12,13], combinatorial
active-site saturation testing (CASTing) [14–16], or the consensus
approach [17] to improve properties of these enzymes, such as
thermostability, enantioselectivity, and substrate specificity. Such
protein engineering techniques require multiple functional and
∗
Corresponding author at: School of Chemical and Biomolecular Engineering and
School of Chemistry and Biochemistry, Parker H. Petit Institute for Bioengineering
and Bioscience, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332-
0363, USA. Fax: +1 404 894 2291.
E-mail address: andreas.bommarius@chbe.gatech.edu (A.S. Bommarius).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.06.014

22
J.K. Blum, A.S. Bommarius / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 21–28
half-life ꢂ1/2, melting temperature Tm, and the temperature of
half-residual activity at 30 min T50 of the purified recombinant
30
Nomenclature
protein. We study the role of the acidic carboxylate cluster through
alanine replacement of the active-site residues. Additionally, we
evaluate the enzyme’s mutability around the active site through
site-directed mutations of residues within 5 Å from the ligand.
Lastly, AEH from X. campestris pv. campestris was employed
to catalyze the kinetically controlled synthesis of the ˇ-lactam
antibiotic ampicillin as shown in Scheme 1. Such a kinetically con-
trolled synthesis raises the issue of selectivity of synthesis versus
the two competing hydrolysis reactions, primary hydrolysis of (R)-
phenylglycine methyl ester ((R)-PGME) and secondary hydrolysis
of ampicillin [3,6,20]. The optimal operating point is reached at the
˛
selectivity enzyme for the antibiotic substrate rela-
tive to the acyl donor
delimits the synthesis/hydrolysis ratio at low nucle-
ophile concentrations, mM
ˇo
ꢀ
delimits the hydrolysis/synthesis ratio at the trans-
formation of an acyl enzyme–nucleophile complex
fraction of unfolded enzyme
˛
CD
kcat
KM
T50
first order catalytic rate constant, s−1
Michaelis constant, mM
30
temperature at which the enzyme half-life is
30 min, ◦C
time point of maximum synthesis product concentration, [P]max
.
ꢁHd
A
Ea
Ed
Tm
kDa
Eapp
e.e.p
enthalpy of deactivation, kJ mol−1
arrhenius pre-exponential factor, s−1
activation energy, kJ mol−1
We determine the synthesis parameters ˛, ˇo, and ꢀ, which can
be used to predict the maximum product yield as defined by Eqs.
(1)–(3) [21–23].
deactivation energy, kJ mol−1
temperature of melting, ◦C
d[Ps]
d[Ph]
ˇo[6 − APA][(R) − PGME] − ˛[Ps(1 + ˇoꢀ[6 − APA])
(1 + ˇoꢀ[6 − APA])([(R) − PGME] + ˛[Ps]
=
(1)
(2)
(3)
kilodalton
apparent enantiomeric ratio
enantiomeric excess of the ampicillin product
(kcat/KM
)
P
s
˛ =
(kcat/KM
)
(R)−PGME
ꢀ
ꢁ
ꢃP
ꢃP
1
=
[6 − APA]
(1/ˇoꢀ) + [6 − APA]
s
ꢀ
h
fully characterized sequences to be fully efficient. Incidentally, X.
citri is inaccessible in the United States as it is regulated as a plant
pathogen causing citrus canker in citrus plants [18], while its cousin
Xanthomonas campestris pv. campestris is available in the ATCC as
ATCC 33913.
Here, Ps is the synthesis product ampicillin, Ph is the hydroly-
sis product (R)-phenylglycine, ꢃP is the initial synthesis rate of
s
ampicillin, and ꢃP is the initial formation rate of the hydroly-
h
sis product (R)-phenylglycine. The differential equation (1) can be
solved numerically to determine [P]max. The Eqs. (1)–(3) have been
successfully used to characterize the kinetically controlled synthe-
sis of ampicillin [21–24].
In
this
investigation,
the
putative
glutaryl
7-
aminocephalosporanic acid (GL-7-ACA) acylase from X. campestris
pv. campestris (gaa) (GI:21113373) was selected for characteriza-
tion. The gaa gene was previously identified in a genome study of
X. campestris pv. campestris [19]; however, the gene has not been
previously isolated nor has the protein been tested for activity. The
gaa gene has a high level of nucleotide alignment (89%) and the
protein features a high level of amino acid identity (93%) with AEH
from X. citri [5]. All key catalytic residues are conserved, including
the catalytic triad, carboxylate cluster, and oxyanion hole (see
Supplemental Materials for BLAST and CLUSTAL W alignment).
In this work, we describe the isolation and cloning of the gene
from genomic DNA, as well as expression and extensive charac-
terization of the putative 7-ACA acylase from X. campestris pv.
campestris. We successfully produce active AEH in E. coli and report
substrate range, kinetic and thermodynamic properties, such as
2. Experimental
2.1. Materials
6-aminopenicillanic acid (6-APA), (R)-phenylglycine (R-
PG), (R)-ampicillin (R-AMP), (R)-phenylglycine methyl ester
hydrochloride ((R)-PGME), (R)-phenylglycine methyl ester
hydrochloride (S-PGME), 7-aminocephalosporanic acid (7-ACA),
7-desacetoxycephalosporanic acid (7-ADCA), cephalexin (CEX),
penicillin G (PenG) all were procured from Sigma Aldrich (St.
Louis, MO). Magic Media was from Invitrogen (Carlsbad, CA),
Ni-NTA Superflow Resin was from Qiagen (Germantown, MD). The
Scheme 1. Synthesis reaction of Ampicillin using AEH.

J.K. Blum, A.S. Bommarius / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 21–28
23
oligonuclueotides for cloning of the gaa gene were purchased from
Eurofins-mwg|operon Biosciences (Huntsville, AL).
native conditions. Next, the pure protein was dialyzed using a
Spectra/Por molecular porous membrane 12–14,000 kDa MWCO in
50 mM phosphate buffer pH 7.0. The purified enzyme had a spe-
cific activity of 59.7 U/mg (total 89.5 U), which resulted in an overall
87% yield of activity for the desired protein. The purified enzyme
was analyzed using 12.5% sodium dodecylsulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) loaded with 15 ␮g of protein per
lane.
2.2. Bacterial strains and plasmids
The genomic DNA encoding the putative glutaryl 7-ACA acylase
(gaa) of Xanthomonas campestris pv. campestris was obtained from
the American Type Culture Collection (ATCC 33913D; Manassas,
VA). E. coli strains BL21 (DE3)pLysS (Promega; Madison, WI) and
XL1Blue were used for expression and cloning, respectively. The
plasmid pET28a (Novagen; Darmstadt, Germany) was utilized as a
cloning and expression vector containing a histidine tag (His6) for
purification. All PCR reactions were performed with recombinant
Pfu polymerase that was purified from E. coli cultivated in our lab.
2.8. Enzyme assays and determination of kinetic constants
Activity of the enzyme was assayed at 25 ◦C by following the
hydrolysis of 20 mM ampicillin in 100 mM phosphate pH 7.0 by high
performance liquid chromatography (HPLC). Before analysis, the
samples were quenched and diluted 10-fold by the addition of HPLC
eluent (5 mM phosphate buffer (pH 3), 300 mg/L sodium dodecyl-
sulfate (SDS), 30% acetonitrile). The initial rates (<10% conversion)
of hydrolysis of all substrates were determined by measuring
product formation by HPLC. To determine kinetic parameters, the
enzyme was incubated with varying substrate concentrations in
the range of 0–30 mM (R)-ampicillin, cephalexin, and penicillin G
each.
2.3. DNA sequencing
The DNA sequences were determined by Eurofins-mwg|operon
Biosciences (Huntsville, AL).
2.4. Cloning of gaa into expression host
For expression of the gaa gene in E. coli, the vector pETXCC
(gaa cloned in pET28) was constructed. Primers were first
used to isolate the gaa gene from the ATCC 33913 DNA, For-
ward primer 5ꢀ-CGCAGTGGCTGGAAGA CATAT-3ꢀ and Reverse
primer 5ꢀ-ATCACCGCAACCACGACCTTTGAC-3ꢀ were used. Forward
primer 5ꢀ-TGCATGCATGCCATGGTTATGCGTCGTCTT GCCGCCTGC-
2.9. Synthesis
Synthesis of (R)-ampicillin and (S)-ampicillin was conducted
at 25 ◦C with 60 mM (R)-PGME and 20 mM 6-APA in 100 mM
phosphate buffer pH 6.2. A racemic mixture of 90 mM (R/S)-
phenylglycine methyl ester hydrochloride was prepared to
determine the enantioselectivity of the reaction. Lastly, synthesis
of cephalexin was performed at 25 ◦C with 60 mM (R)-PGME and
20 mM 7-ADCA. Before analysis the samples were quenched and
diluted 10-fold by the addition of HPLC eluent. The analysis was
conducted with HPLC.
3ꢀ
ACCGGCAGACTGATG-3ꢀ with restriction sites Nco
Hind III, underlined, were used to incorporate the gaa gene
into pET 28 vector system.
second reverse primer 5ꢀ-
and
reverse
primer
5ꢀ-CGGCGGCCCAAGCTTTCAACGC
I
and
a
A
CGGCGGCCCAAGCTTACGCACGGCAGACTGATGTAG-3ꢀ was used to
incorporate the C-terminal 6X his tag in the pET 28 vector for ease
of purification. After denaturation of the DNA, the amplifications
were completed in 30 cycles of 30 s at 98 ◦C, 1 min at 52 ◦C, and
4 min at 72 ◦C. Products and vector were digested with NcoI and
HindIII and ligated. The ligation mixture was used to transform
chemically competent E. coli BL21(DE3)pLysS (Cmr). The construct
was confirmed by sequencing.
2.10. HPLC analysis
All analysis was conducted using high performance liquid chro-
matography complete with a Shimadzu-LC-20AT pump, Beckman
Coulter Ultrasphere ODS 4.6 mm × 25 cm column, and SPD-M20A
prominence diode array detector (PDA) monitored at 215 nm. The
mobile phase is isocratic at 1.0 mL/min and contains 30% ace-
tonitrile and 70% 5 mM phosphate buffer with 300 mg/L sodium
dodecylsulfate (SDS) (pH 3), method adapted from Gabor and
Janssen [24]. All components, (R)-PG, (R)-PG, 6-APA, (R)-PGME,
(S)-PGME, (R)-ampicillin, (S)-ampicillin, analyzed on the HPLC
with >95% mass balance closure. The (R)-ampicillin and (S)-
ampicillin diastereomeres can be separated on the HPLC, however,
the stereoisomers of RPG/SPG and (R)-PGME/(S)-PGME co-eluted.
Enantiopurity of the product (e.e.p) was determined using the (R)-
Amp and (S)-Amp concentrations.
2.5. Site-directed mutagenesis
Variants D208A, E309A, and D310A and double and triple
mutants E209A/D310A, D208A/E309A/D310A were generated
using overlap PCR and ligated into the pET 28 vector system. All con-
structs were confirmed by sequencing. Other active-site variants
were made using the QuikChange® approach.
2.6. Recombinant overexpression of the AEH
A 5-mL culture was inoculated with a single colony, grown
overnight (15 mL test tubes, 30 ◦C, 250 rpm) and subcultured
into (1:40 [v:v]) in Invitrogen MagicMediaTM (Carlsbad, CA) E.
coli expression medium supplemented with 30 ␮g/mL kanamycin,
30 ␮g/mL chloramphenicol (Cm) (shake flask with baffles, 30 ◦C,
200 rpm). After 6 h, the cells were incubated for 24 h at 25 ◦C.
2.11. Circular dichroism (CD)
All analysis was conducted using a JASCO CD J-810 with quartz
cuvettes. Thermal scans ranged between 5 and 95 ◦C with a scan
rate of 1 ◦C/min. Data was analyzed to determine ꢁH and Tm as
described in Greenfield [25].
2.7. Isolation of recombinant putative glutaryl 7-ACA acylase
(gaa) from E. coli
3. Results and discussion
The cells were harvested by centrifugation and resuspended
in 15 mL of cold lysis buffer (50 mM potassium phosphate (pH
8.0), 10 mM imidazole, and 300 mM sodium chloride) per 1 g of
wet cell weight. The clarified cell lysate had a specific activity
of 1.6 U/mg (total 103 U). The mixture was then sonicated and
batch-purified as per the Ni-NTA His-Bind resin protocol under
3.1. Cloning, expression, and purification
The complete 1914 bp gaa gene of X. campestris pv. campestris
was isolated from the genomic DNA via PCR with primers just out-
side of the desired gene. The gene was cloned into pET28 with

24
J.K. Blum, A.S. Bommarius / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 21–28
Table 1
Kinetic constants for the AEH from X. campestris pv. campestris.a.
Substrate
kcat (s−1
)
KM (mM)
kcat/KM (s−1 mM−1
)
(R)-ampicillin
Penicillin G
Cephalexin
(R)-PGME
72.5
n.d.b
200
982
1.1
64.5
n.d.b
95
n.d.b
2.2
3.7
265
a
All reactions performed at 25 ◦C and pH 7, as described in Section 2.
n.d., not detected.
b
an optimum pH of 6.8 was found (Supplemental Fig. S-1). Fig. 2A
illustrates the temperature dependence of the AEH activity for the
hydrolysis of ampicillin. The optimum temperature was found to be
25 ◦C. Between 6 and 25 ◦C, the activity increased with an activation
energy of 56.7 kJ mol−1 (see Supplemental Material for Arrhenius
fits). CD scans (Fig. 2B) were performed on the pure protein to deter-
mine the melting temperature (˛CD = 0.5) of the protein. Using a
two-state model, the melting temperature Tm was determined to
be 32.4 ◦C, the enthalpy of deactivation ꢁHd was calculated with
residual activity data between 25 and 40 ◦C to be 174 kJ mol−1 (R2
of 0.99). The protein was also evaluated by incubating at a range of
temperatures for 30 min, resulting in a T5030 value of 27 2 ◦C, fur-
ther illustrating that the enzyme is not very thermostable (Fig. 2C).
As the rate of deactivation is not clearly first order at 30 ◦C, we just
report the observed half-life of the protein: 5 min (Fig. 2D). This low
thermostability potentially can be associated with subunit dissoci-
ation [27,28]. Additionally, it is likely that the low thermostability
of this protein hinders the ability for the protein to tolerate muta-
tions, which tend to further decrease thermostability, as suggested
by both Tokuriki and Tawfik [30] and Bloom and Arnold [29]. Fur-
thermore, the deactivation and unfolding both occur in a broad
temperature range between 25 and 40 ◦C, indicating that the pro-
tein unfolds in a fairly non-cooperative manner.
Fig. 1. Protein gel of the expressed AEH protein from X. campestris pv. campestris.
Lane 1: pETXccH protein lysate, Lane 2: pETXccH unbound fraction, Lane 3: pETXccH
Wash 1, Lane 4: pETXccH Wash 2, Lane 5: pETXccH eluent. The desired protein band
is approximately 68 kDa.
and without the C-terminal 6X histidine tag, resulting in an active
construct that is designated pETXcc and pETXccH, respectively, as
described in Section 2 . The gene encodes a 637 amino acid polypep-
tide; the gene sequence was confirmed by DNA sequencing. The
first 22 amino acids encode a signal peptide as predicted by the
SignalP 3.0 Server [26], thus resulting in an active peptide subunit
of 615 amino acids and a calculated molecular weight of 68 kDa.
These constructs were transformed into E. coli BL21(DE3)pLysS
cells. Overall expression is 3% of the total soluble protein content,
similar to expression levels previously reported for AEHs [5,6,9].
Activities of soluble lysate from pETXcc and pETXccH were com-
pared indicating that the histidine tag did not impact expression of
the plasmid, confirming previous work on the AEH from A. turbidans
[5,6,9]. Pure protein was obtained through immobilized metal ion
affinity chromatography IMAC purification on Ni-NTA of pETXccH
as described in Section 2. The SDS-PAGE protein band at 68 kDa is
the desired AEH protein (Fig. 1).
3.4. Mutability of active site and surrounding residues
The residues of the carboxylate cluster (Fig. 3A) were
each mutated to alanine (D208A, E309A, D310A). Addition-
ally, the double mutant (E309A/D310A) and triple mutant
(D208A/E309A/D310A) were constructed. These mutants were all
successfully expressed in BL21(DE3)pLysS as observed by overex-
pression of soluble protein (SDS-PAGE gels not shown). The single
variants D208A and E309A both destroyed 100% of the wild-type
(WT) activity toward ampicillin and cephalexin, and variant D310A
reached only 4% ampicillin conversion after 24 h of incubation at
25 ◦C. Both the double mutant and triple mutant resulted in no
detectable activity. Despite replacing all three residues that are
required for recognition of the ˛-amino group on ampicillin with
alanine, none of the mutants showed activity toward penicillin G,
which lacks the ˛-amino group, thus indicating that the carboxy-
late cluster residues are critical for both the activity and substrate
specificity of this enzyme.
Additional single point mutations were constructed around the
active site of the enzyme: Y82A, S174A, Y175 (A,R,D,C,H,L,S,V),
D207(A,L,N), D208(L,N), W209A, Y222A, D307N, E309(L,Q), and
D310(L,N). These residues are all within 5 Å of the active site. S174,
D307, H340 (not mutated) form the catalytic triad, Y82A, Y175A are
part of the oxyanion hole, and D208, E309, D310 form the carboxy-
late cluster. Barends et al. found that a mutation at position Y206A
(AEH from X. campestris pv. campestris residue Y175A) decreased
the enzyme activity but led to improved synthesis properties [6].
The functions of W209 and Y222 are unknown, but are expected to
provide bulk to the enzyme pocket and be involved in pi–pi stacking
with the substrate. It was expected that some of these mutations
would dramatically affect the enzyme activity; however, previous
3.2. Hydrolysis kinetics
The AEH family is specific for esters and amides bearing an ˛-
amino group, however, it is able to accept both cephalosporins
and penicillins as the ˇ-lactam moiety. For this reason, a range
of substrates that include these combinations was tested under
hydrolysis conditions. The results are summarized in Table 1.
Indeed, our protein from X. campestris is active only on ˛-amino
containing ˇ-lactam antibiotics and as thus did not accept peni-
cillin G. The kcat and KM values of ampicillin hydrolysis at 25 ◦C and
pH 6.8 were 72.5 s−1 and 1.1 mM, respectively.
3.3. pH and temperature optima
The hydrolysis activity of AEH on ampicillin as a function of the
pH value and buffer system was studied between pH 4 and 10;

J.K. Blum, A.S. Bommarius / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 21–28
25
Fig. 2. (A) The temperature profile for the hydrolysis of ampicillin using the AEH from X. campestris pv. campestris. An Arrhenius fit of the temperature activation energy
(Ea = 57 kJ mol−1, A = 7.3E+11 s−1, R2 = 0.95) and deactivation constants (Ed = −82 kJ mol−1, A = 3.4E−14 s−1, R2 = 1.0). Only the first four deactivation points were considered in
the fit, initial activity at >35 ◦C is difficult to quantify since the deactivation occurs in <1 min. (B) CD thermal scan result fit to a two-state deactivation model, resulting in
a calculated Tm = 32.4 ◦C. (C) Residual activity of AEH after incubation at reported temperature for 30 min, protein was immediately quenched on ice prior to determining
residual activity at 25 ◦C as described in Section 2. (D) Residual activity at 25 ◦C of AEH after incubation at 30 ◦C at reported time.
work with serine hydrolases suggest that only the mutations on
the catalytic serine, S174, and histidine, H340, should destroy all
100% activity of the enzyme. In contrast to expectations, with the
exception of Y175A and D310N, none of the variants exhibited any
ampicillin conversion after 24-h incubation at 25 ◦C. To test if the
lack of AEH thermostability masks specificity toward the variants
listed above, an additional reaction was conducted at 17 ◦C; higher
ampicillin conversions were achieved for both Y175A (59%) and
D310N (40%) after 24 h under these conditions. The other single
variants still did not exhibit any conversion, thus indicating the crit-
ical nature of all of these active-site residues, including carboxylate
cluster residues D208 and E309, for the activity of this enzyme.
Lastly, to investigate the role of the conserved active-site
residues in homologs, we compared X. campestris AEH to Rhodococ-
cus sp. cocaine esterase (RhCocE) [31] and J1 glutaryl 7-ACA acylase
(J1) [32]. Both the RhCocE and the J1 enzyme do not require an
˛-amino group in the substrate (Table 2). Both the sequence and
crystal structures of AEH pdb:2B4K and RhCocE pdb:1JU3 were
aligned using CLUSTAL W and superimposed using PyMol (DeLano
Scientific), respectively (see Supplemental Material). The PyMol
alignment was anchored on the position of the catalytic triad; from
this alignment the alpha carbon atoms superimposed with an RMS
of 0.6 Å (Fig. 3A). In both RhCocE and J1 the catalytic triad and
oxyanion hole are conserved, while different amino acid residues
Fig. 3. A. PyMol representation of the active-site residues from the AEH from A. turbidans shown with (R)-PG bound, pdb 2b4k [22] aligned with the RhcocE. The proteins
were aligned using PyMol software using the alpha carbons of the catalytic triad (Xcc SER-174, Xcc HIS-340, and Xcc ASP-307) to an RMS of 0.6 Å. The residue number reflects
the numbering for X. campestris pv. campestris (XCC) and for the RhcocE. B. RosettaBackrub model [33] for mutation Asp207Asn, the top 10 structures are shown (blue) the
wild-type structure is shown in green. The position of the Asp208 side chain is repositioned away from the ˛-amino of (R)-phenylglcyine, which impacts the polar interactions
between (R)-PG and Asp208. (For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this article.)

26
J.K. Blum, A.S. Bommarius / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 21–28
Table 2
cephalexin. While D207 does not directly interact with the ˛-amino
substrate, as the ꢀ-carbon of D207 is >4 Å from the ˛-amino moiety
on the substrate, the side chain D207 has polar interactions with
the backbone of several second-shell residues (W312, G313, I201,
D202). These backbone interactions are important in the position-
ing of its adjacent residue D208 that interacts with the substrate
as part of the carboxylate cluster. This scenario was further con-
firmed using a RosettaBackrub model [33] of the D207N mutation.
In this model not only were the side chain to backbone interac-
tions of D207 eliminated, the position of the ꢀ-carbon of the D208
residue was moved 0.8–1.5 Å from the original position, which may
disrupt the salt bridge formed with the substrate (Fig. 3B). These
results suggest that the entire group of acidic residues near the
active site, D207, as well as D208, E309, and D310, are critical for
protein function.
Alignment of active-site residues of X. campestris pv. campestris AEH, RhCoCE, and
J1 acylase.
X. campestris pv.
campestris
RhCocEa
J1 acylasea
Comment
Y82
Y44
S117
Y84
S152
Y153
E184
V185
F186
Oxyanion hole
Active serine, catalytic triad
Oxyanion hole
S174
Y175
D207
D208
W209
Y222
D307
E309
D310
H340
Y118
A149
P150
W151
W166b
D259
–, L407c
F261
W200b
D291
–, T470c
L293
Larger aromatic
Catalytic triad
H287
H336
Catalytic triad
a
Residue numbering includes the signal sequence of both RhCocE and J1 acylase.
The CLUSTAL W sequence alignment indicates that G165 and S199 should align
b
with Y222, however, in the 3D homology model (Supplemental Material), it is clear
that the residues W166 and W200 (based on homology) properly align at this site.
3.5. Synthesis of ampicillin
c
While there is a gap in the sequence alignment, 3D alignment using PyMol
indicates that these residues fill the space occupied by E309 in X. campestris pv.
campestris.
(R)-ampicillin was synthesized through enzymatic acylation
of (R)-PGME with 6-APA in purely aqueous system as shown in
Scheme 1 and Fig. 4A. This reaction is coupled with two enzyme-
catalyzed side reactions, primary hydrolysis of (R)-PGME to (R)-PG
and methanol and secondary hydrolysis of (R)-ampicillin to (R)-
PG and 6-APA. The results of the synthesis reaction are shown
in Fig. 4. At [6-APA] = 20 mM and [(R)-PGME] = 60 mM, the maxi-
mum product concentration [P]max achieved was ∼10 mM, or 50%
conversion with respect to 6-APA. The reaction stalls 40 min with
substrate still available: concentrations at 40 min are 10 mM (AMP),
10 mM (6-APA), 35 mM ((R)-PG), and 15 mM ((R)-PGME). Even
after 24 h under these conditions the product remained in solution
at 10 mM and no secondary ampicillin hydrolysis was observed.
Enzyme activity has been confirmed for the duration of the synthe-
sis; thus, the lack of conversion is not due to poor enzyme stability.
are found at AEH positions D207, D208, W209, Y222, E309, and
D310. Positions W209 and Y222 are replaced by different aromatic
residues suggesting that these changes compensate for size vari-
ability in the substrate. In the sequence alignment of X. campestris
pv. campestris AEH and RhCocE, a gap appears at the E309 position.
However, in the PyMol superposition of RhCocE on AEH, it appears
that loop residue RhCocE L407 or the homologous J1 T470 residue
fills the corresponding space in the binding pocket with a small
non-polar amino acid. At position D207, the RhCocE contains an
alanine while J1 acylase contains a glutamate.
Our X. campestris pv. campestris AEH variants D207A, D207N,
and D207L did not show significant activity toward ampicillin or
Fig. 4. (A) Kinetically controlled synthesis of (R)-ampicillin with AEH from X. campestris pv. campestris starting with 20 mM 6-APA and 60 mM R-PGME (B) Kinetically
controlled synthesis of (R)-ampicillin employing AEH from X. campestris pv. campestris starting with 20 mM 6-APA and 60 mM S-PGME (C) Synthesis of (R/S)-ampicillin
starting with 20 mM 6-APA and 90 mM racemic PGME. (D) Plot of the change in enantioselectivity of the reaction over time, when starting with racemic PGME.

J.K. Blum, A.S. Bommarius / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 21–28
27
low formation of the hydrolytic by-products from both primary
and secondary hydrolysis. The ˇo and ꢀ values for wild-type PGA
ampicillin synthesis range from ˇo = 0.06 to 0.50 mM−1 and 1/ꢀ = 6
to 16 [23,24,35], with mutant PGA PAS2 (TM33) reported to have
ˇo = 10.2 mM−1 and 1/ꢀ = 286 for ampicillin synthesis [24]. To the
best of our knowledge, ˇo and ꢀ values have not previously been
calculated for this class of enzymes.
The kinetic parameters are able to closely estimate Pmax when
fit employing Eqs. (1)–(3) (Fig. 5). While the model appropriately
tracks the raw data (Fig. 5), it should be noted that when (R)-PGME
is in excess (20:45, 20:60) the model does not predict the lack
of secondary hydrolysis when the (R)-PG concentration reaches
∼32 mM, as observed by the clusters of points on the 20:45 and
20:60 curves. This finding suggests an inhibition effect of (R)-PG at
this concentration.
4. Conclusions
Fig. 5. Plot of [RPG] versus [AMP] results for six synthesis reactions at various
[6APA]o:[RPGME]o ratios and concentrations (20:20 mM, 33:20 mM, 45:20 mM,
60:20 mM, 20:33 mM; 20:45 mM; 20:60 mM), fit to the model equation (Eq. (1))
with parameters ˛ = 0.25, ˇo = 42.8 M−1, and ꢀ = 0.23. The experimental results are
shown by the data points, while the model fits are represented by the lines.
The AEH from X. campestris pv. campestris strain ATCC 33913
displayed comparable levels of specific activity and improved syn-
thetic properties compared to AEH from X. citri and possibly also
to AEH from A. turbidans, as evidenced by its lower ˛-value, ˇo
and clearly favorable compared to the PGA derived enzymes, and
ꢀ values comparable to wt-PGA. The enzyme’s unique ability to
catalyze the ˇ-lactam synthesis with minimal secondary hydrol-
ysis in purely aqueous solutions renders this enzyme an even
more interesting target for ˇ-lactam synthesis. The most commonly
industrially used enzyme currently employed for this reaction,
penicillin G acylase (E.C. 3.5.1.11), exhibits significantly more sec-
ondary hydrolysis, unless it is reacted in a biphasic system or highly
concentrated solutions [3,4,21].
The current understanding of the carboxylate cluster focuses
on its involvement in the recognition of the ˛-amino group on
the substrate, thus positioning the substrate for catalysis [5]. We
argue that our results demonstrate that D207 should be regarded
as part of the critical residues required for ˛-amino group recog-
nition, just as D208, E309, and D310. Surprisingly, a single alanine
replacement of any of the critical residues totally suppressed or at
least significantly decreased the wild-type activity of the enzyme.
We hypothesize that this observation is due to the need for a
highly electron-deficient substrate carbonyl carbon in addition to
the requirement of the ˛-amino group to be hydrogen-bonded to
the carboxylate residues. Replacement of the negatively charged
residues with alanine was insufficient to shift the substrate range to
antibiotics that lack the ˛-amino group. We therefore conclude that
the carboxylate cluster is critical for both recognition and catalysis.
We have successfully expanded the available repertoire of AEHs.
The lack of mutability around the AEH active site suggest that these
proteins are highly evolved for their substrate specificity toward
˛-amino carboxylic acid electrophiles, as there are many residues
associated with its substrate specificity. The enzyme has very good
synthetic properties toward the substrate and would be useful for
industrial applications.
Furthermore, secondary hydrolysis can occur when using <40 mM
(R)-PGME as the substrate (Fig. 5). Similar results for secondary
hydrolysis were obtained for cephalexin synthesis with partially
pure enzyme isolated from X. citri (non-recombinant), when using
a 20 mM (7-ADCA):60 mM ((R)-PGME) ratio [34]. Employing X.
campestris pv. campestris AEH, (S)-ampicillin was synthesized anal-
ogously to the (R)-enantiomer (Fig. 4B). The initial synthesis rate
of (S)-ampicillin was 0.31 mM min−1 over the linear portion of the
synthesis curve, only one seventh of the (R)-ampicillin synthesis
rate of 2.0 mM min−1, indicating the enzyme’s preference for the
(R)-substrate. Enantioselectivity was also tested in the compet-
ing synthesis of both (R)- and (S)-ampicillin (Fig. 4C) employing a
starting ratio of 90 mM ((R/S)-PGME): 20 mM (6-APA). While the
enzyme has a preference for (R)-ampicillin, the resulting enan-
tiomeric excess e.e.p of the ampicillin product was only 22% at
steady state (Fig. 4D), resulting in an apparent enantiomeric ratio
Eapp of only 2.4 (Eq. (4)). Higher overall yields were observed when
starting with racemic PGME, with [P]max at 14 mM resulting in 70%
conversion of the 6-APA substrate.
ꢂ
ꢃ
1 − x(1 + e.e.p)
1 − x(1 − e.e.p)
Eapp = ln
(4)
where x = degree of conversion.
Kinetic parameters ˛, ˇo and ꢀ were determined to fully char-
acterize the synthetic properties of this enzyme. ˛-Values for
the synthesis of ampicillin with the wild-type AEHs range from
˛ = 0.15–1.09 for A. turbidans to ˛ = 2.2 for X. citri, calculated from
previously reported data [5]. Reported ˛-values for the PAS2 peni-
cillin G acylases range ˛ = 7.6–16.4 for (R)-PGME [24]. The ˛-value
for the AEH from X. campestris pv. campestris was calculated from
the initial hydrolysis data (Table 1) to be 0.25, at the low end of the
reported range. A low ˛ is desirable as it is an indication for the
enzyme’s specificity for the synthesis substrate ((R)-PGME) over
that of the acyl donor, whereas a high ˛ would indicate high sec-
ondary hydrolysis rate. The nucleophilic reactivity is defined by
the ˇo and ꢀ values. ˇo and ꢀ were determined from a non-linear
regression of a plot of [6-APA]o, which ranged from 20 to 60 mM,
at constant [(R)-PGME]o concentration of 20 mM, versus the ini-
tial synthesis:hydrolysis ratio observed during synthesis reactions.
The parameters were calculated to be ˇo = 42.8 M−1 and ꢀ = 0.23
with an R2 of 0.99 (see Supplemental Material). It is preferable that
both ˇo and 1/ꢀ (1/ꢀ = 4.3) values to be high [22,24] to indicate
Acknowledgements
The authors gratefully acknowledge support from the National
Institute of Health (Grant # 5R01AI064817-02). The authors would
like to thank Dr. Javier Chaparro-Riggers, Dr. Baraheh Azizi, Dr.
Mélanie Hall, Patrick Garrett, Evelina Ponizhaylo, and Michael D.
Ricketts for their guidance and assistance in the preparation of this
work. We also thank Prof. Dick Janssen of the University of Gronin-
gen (Groningen, Netherlands) for helpful discussions. The authors
would also like to thank Aaron Engelhart in the lab of Dr. Nick Hud at
Georgia Tech for help with the CD instrumentation. J.K.B. gratefully
acknowledges funding by a NSF Graduate Research Fellowship.

28
J.K. Blum, A.S. Bommarius / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 21–28
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