E. Rosini et al. / Journal of Molecular Catalysis B: Enzymatic 76 (2012) 52–58
53
substituent can also be quite different from a -lactam skeleton
[8,19,20].
equation or modified to account for a substrate inhibition effect
[25,26].
2.4. Screening for acylase activity on different substrates
In this work we compare the substrate preference of a GA widely
used in industry (glutaryl acylase commercialized by Recordati
approach based on error-prone PCR mutagenesis, site-saturation
mutagenesis and site-directed mutagenesis driven by a molecu-
lar modelling analysis (VAC, both the wild-type and H296S-H309S
variant) [21].
The acylase activity on different compounds was assayed by a
semi-quantitative colorimetric screening procedure based on the
reaction of pDMAB with the free NH2-group produced starting from
different substrates. The extinction coefficient of each Schiff’s base
is different depending on the substrate used, this affecting both
the precision and sensitivity of the measurement. In details, 50 L
of enzyme and 50 L of substrate (5 or 50 mM final concentra-
tions) were transferred to wells of a 96-well plate and incubated
at 25 ◦C for 10 min. The reaction was stopped by adding 100 L of
stop solution (30% acetic acid); then 50 L of 0.35% (w/v) pDMAB
in methanol was added. After 10 min of incubation at 25 ◦C, the
absorbance value at 405 nm was measured by a microtiter plate
reader (Sunrise, TECAN) and compared with the value obtained
with Gl-7-ACA as reference. In addition to the substrates reported
in Table 1, the following compounds have been tested: glutaryl-
d-alanine, -phenylalanine, -phenylglycine; glutaryl-l-alanine, -
phenylalanine, -phenylglycine; methylesters of glutaryl-d-alanine,
-phenylalanine, -phenylglycine, -leucine, -tryptophan, -alanine;
methylesters of glutaryl-l-alanine, -phenylalanine, -phenylglycine,
-leucine, -tryptophan, -alanine and glutaryl-glycine methylester;
glutaryl p-nitrobenzyl amide; glutaryl 1-phenylethyl amide; glu-
taryl benzyl amide; glutaryl cyclobutyl amide; glutaryl cyclopentyl
amide; glutaryl cycloesyl amide; succinyl benzyl amide.
2. Experimental
2.1. Enzymes
Commercial glutaryl acylase GAR was a gift from Recordati S.p.A.
(Opera, MI, Italy); its specific activity on Gl-7-ACA is 2.3 U/mg pro-
tein [20]. Recombinant VAC proteins (wild-type and H296S-H309S
VACs) were produced as stated in [22,23]. From 1 L of fermenta-
tion broth, 55 and 77 mg of wild-type and H296S-H309S VACs were
produced with a specific activity of 20.2 and 2.4 U/mg protein on Gl-
7-ACA as a substrate, respectively. The final enzyme preparations
were equilibrated in 20 mM potassium phosphate buffer, pH 8.0.
The amount of protein was estimated by the absorbance at 280 nm
using the molar extinction coefficient of 110 mM−1 cm−1
.
2.2. Substrate synthesis
2.5. Hydrolytic reactions (pH-Stat)
Cephalosporanic amides were prepared by reaction with a suit-
able anhydride as described in our previous paper [18]. Briefly,
the substrate – either 7-ACA, 7-aminodesacetoxycephalosporanic
acid (7-ADCA), 7-Z-aminocephalosporanic acid or 3 chloro-7-
aminodesacetoxycephalosporanic acid, 5 mmol – was dissolved in
20 mL of 1 M NaHCO3, while the anhydrides (1 equiv.) were diluted
in 5 mL acetone. The two solutions were mixed and let to react for
at least 3 h (TLC: n-BuOH:AcOH:H2O = 6:2:2). Acetone was evap-
orated, the water solution was acidified to pH 1.5 with 1 M HCl
and extracted three times with 100 mL AcOEt. The organic layer
was evaporated and the solid residue was washed on a Buchner
funnel with 20 mL AcOEt and dried. Amino acidic derivatives were
prepared by a similar procedure, but dissolving both the substrate
(5 mmol) and the anhydride (1 equiv.) in anhydrous dioxane (30
and 5 mL, respectively), and maintaining the reaction under stirring
at 50 ◦C for at least 24 h. After evaporation of dioxane, the prod-
uct was redissolved in 20 mL of water and recovered as described
previously.
Hydrolytic reactions were monitored at 20 ◦C using a 718 STAT
Titrino automatic titrator (Metrohm Ltd.). In a total volume of
10 mL, 50 mM substrate dissolved in H2O and a suitable amount
of GAs were stirred while maintaining the pH at a constant value of
8.0 by adding 0.1 M NaOH. The rates of hydrolysis were calculated
from the amount of NaOH solution added in the time unit. Relative
activity on various substrates was compared with that observed for
Gl-7-ACA, fixed as 100%.
3. Results
3.1. Analysis of substrate preference
A preliminary screening of the activity of wild-type and H296S-
H309S VAC enzymes on a number of compounds was carried out
by the pDMAB-based colorimetric assay using a 96-well plate. For
these measurements two amounts of enzyme (0.75 and 7.5 g for
wild-type, 19 and 190 g for H296S-H309S VAC) and two substrate
concentrations (5 and 50 mM) were employed. At 5 mM substrate
concentration, both VAC enzymes showed a negligible activity on
amino acid derivatives of glutaric acid, as well as on the corre-
sponding methyl esters or on glutaryl amides of cyclic amines (not
shown). On the contrary, an increase in absorbance at 405 nm was
observed with most of the tested cephalosporanic derivatives: the
relative activity values – vs. Gl-7-ACA as a reference substrate, fixed
as 100% – are reported in Table 1. At 50 mM substrate concentra-
tion a similar pattern was observed, the main exception being a
substantial increase in activity on pentanoyl-7-ACA and glutaryl-3-
H296S-H309S VAC variant. The low activity determined at 5 mM
CephC with the latter enzyme (≈15% as compared to Gl-7-ACA) is
mainly related to the comparatively high Km value for this sub-
strate, see below and [21]. For some of the compounds tested, the
activity values determined at 50 mM substrate concentration were
2.3. Enzymatic activity and kinetic measurements
The standard activity assay was based on the hydrolysis of Gl-7-
ACA to 7-ACA and the subsequent formation of a yellow Shiff’s base
(with a maximum of absorbance at 415 nm, ε = 0.635 M−1 cm−1
)
by the reaction of p-4-dimethylaminobenzaldehyde (pDMAB) and
the primary amino group of 7-ACA [21,24]. One unit of acylase is
defined as the amount of enzyme that converts 1 mol of sub-
strate per minute under the assay conditions. In details: 0.1 mL
of enzyme was mixed with 0.1 mL of 1% (w/v) Gl-7-ACA at pH
8.0, and incubated for 10 min at 25 ◦C. The reaction was stopped
by adding 0.6 mL of 20% acetic acid, and then 0.133 mL of 0.5%
(w/v) pDMAB (dissolved in methanol) was added and the mixture
was incubated for further 10 min at 25 ◦C, before measuring the
absorbance at 415 nm. The kinetic parameters were determined
similarly using a fixed amount of enzyme and different substrate
concentrations (0–150 mM): activity vs. substrate concentration
data were analyzed according to the classical Michaelis–Menten