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S. Del Prete et al. / Bioorg. Med. Chem. 24 (2016) 220–225
appropriate amount of PEPC preparation. The reaction was started
3. Conclusions
by the addition of PEP. One unit of PEPC will oxidize one micromole
of NADH per minute at 25 °C. CA activity was assayed as described
in previously published procedures.20–22 Briefly, an Applied Photo-
physics stopped-flow instrument has been used for assaying the CA
catalyzed CO2 hydration activity. Bromothymol blue (at a concen-
tration of 0.2 mM) has been used as indicator, working at the
absorbance maximum of 557 nm, with 10–20 mM Tris (pH 7.5
We constructed an in vitro enzymatic system with mesophilic
and thermophilic enzymes, which is constituted by a recombinant
PEPC from the thermophilic cyanobacterium T. elongatus and
mesophilic or thermophilic bacterial CAs, with the purpose of con-
verting CO2 into a C4-dicarbossilic acid (OAA) of great potential for
use in industrial/pharmacologic applications. We also evaluated
the potential use of these CAs in improving OAA production. Our
results demonstrated that: (1) all the CAs tested were able to
enhance OAA production catalyzed by PEPC; (2) VchCA (a-CA
class) was the most efficient in improving OAA production; (3) Tel-
PEPC was thermostable and thermoactive; (4) TelPEPC and SazCA
(thermoactive/thermostable bacterial a-CA) coupled together were
able to produce OAA even if exposed to temperatures up to 60 °C,
suggesting a pivotal role in biotechnological processes. The enzy-
matic conversion of carbon dioxide, which is inspired by the CO2
metabolic process in cells, offers a green and potent alternative
for efficient CO2 capture, sequestration, and utilization.
for
a- and c-CA; pH 8.3 for b-CA) as buffer, and 20 mM Na2SO4
for maintaining constant the ionic strength (this anion is not inhi-
bitory and has a KI > 200 mM against this enzyme), following the
initial rates of the CA-catalyzed CO2 hydration reaction for a period
of 10–100 s, at 25 °C. The CO2 concentrations ranged from 1.7 to
17 mM for the determination of the kinetic parameters and inhibi-
tion constants. For each measurement at least six traces of the ini-
tial 5–10% of the reaction have been used for determining the
initial velocity. Typically the first 10 s of the traces were used for
determining the initial rates, which has been done by linear fitting
with a program furnished by Applied Photophysics. The CO2 con-
centrations ranged from 1.7 to 17 mM for the determination of
the kinetic parameters and inhibition constants. The uncatalyzed
rates (which needed times of around 30 s in our assay conditions)
were determined in the same manner and subtracted from the
total observed rates.
4. Experimental
4.1. Cloning, protein expression, and purification of TelPEPC
The identification of T. elongatus PEPC (TelPEPC) was performed
enolpyruvate carboxylase and cyanobacteria’ as keyword. The
Eurofins Genomics Company, specialized in gene synthesis,
designed the synthetic TelPEPC gene and directly inserted it into
the expression vector pET-15b. Competent E. coli cells (Arctic
Express DE3) were transformed with pET-15b/TelPEPC vector,
grown at 30 °C, and induced with 1 mM IPTG. After IPTG induction,
cells were grown at 20 °C for further 6 h, then harvested and dis-
rupted by sonication at 4 °C. Following centrifugation at 12,000g
for 30 min, the supernatant was loaded onto a HIS-TRAP FF crude
affinity column (GE Healthcare). The enzyme was eluted with
250 mM imidazole. At this stage of purification the enzyme was
about 70% pure.
4.4. SDS–PAGE
Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophore-
sis (PAGE) was performed according to Laemmli24 using 12.5% gels.
4.5. OAA assay
Quantification of OAA produced by fixation of CO2 with PEP was
performed using the Oxaloacetate Assay Kit by Sigma–Aldrich. In
this kit, oxaloacetate concentration is determined by a coupled
enzyme assay, which results in a colorimetric (570 nm) product,
proportional to the OAA present. Briefly, TelPEPC (5 mU), with or
without the amount of the tested CAs (see Figs. 3 and 4), was incu-
bated in a final volume of 50 lL reaction buffer (50 mM Tris–HCl,
4.2. Cloning, protein expression, and purification of bacterial
CAs
pH 8, 1 mM MgCl2, 6 mM PEP) in order to allow the carboxylation
of PEP. HCOꢀ3 was not added in the assay, but it originated from the
ambient CO2 in reaction buffer. After the reaction time indicated in
The GeneArt Company designed the synthetic V. cholerae gene
encoding the b-CA, containing 4 base pair sequences (CACC) neces-
sary for directional cloning at the 50 end of the VchCAb gene. The
fragment was subsequently cloned into the expression vector
pET100/D-TOPO (Invitrogen). Competent E. coli BL21 (DE3) cells
were transformed with pET100/D-Topo/VchCAb vector, grown at
37 °C, induced with 1 mM IPTG and grown for 4 h. After additional
growth for 4 h, cells were harvested and disrupted by sonication at
4 °C. Following sonication, the sample was centrifuged at 1200g for
30 min and the supernatant loaded onto a His-select HF Nickel
affinity column (GE Healthcare). The VchCAb was eluted with
0.02 M phosphate buffer (pH 8.0) containing 250 mM imidazole
and 0.5 M NaCl. At this stage of purification the enzyme was at
least 95% pure. VchCA, PgiCA, and SazCA were obtained according
to previously published procedures.20–22
Figures 3 and 4, the OAA assay buffer of the kit (50 lL) was added
and the resulting mix incubated for 30 min, protected from light.
The amount of OAA produced was determined by measuring
OD570. An appropriate OAA standard curve was set up each time
the assay was run. In the thermal inactivation experiment
(Fig. 5), Tel PEPC and SazCA were incubated at 25, 40, 50, 60, and
70 °C for 15 min, cooled on ice, and then assayed as described
above.
References and notes
4.3. PEPC and bacterial CAs activity
PEPC activity was routinely measured in a coupled enzyme
assay using malate dehydrogenase by spectrophotometrically
monitoring the decrease in A340 resulting from NADH oxidation.23
The assay mixture consisted of 80 mM Tris-sulfate, pH 8.5,
10.5 mM MgSO4, 0.21 mM ß-NADH, 10 mM NaHCO3, 10% (v/v)
dioxane, 10 mM DTE, 1 mM PEP, 6 U malic dehydrogenase, and