Recombinant thermoactive phosphoenolpyruvate carboxylase (PEPC) from Thermosynechococcus elongatus and its coupling with mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the conversion of CO2 to oxaloacetate

Article abstract of DOI:10.1016/j.bmc.2015.12.005

With the continuous increase of atmospheric CO₂ in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO₂ into useful chemicals could be a good strategy to both decreasing the CO₂ concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO₂ mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO₂ to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO₂ into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO₂ into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to α-, β-, and γ-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO₂ hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 °C, suggesting a possible role of the two coupled enzymes in biotechnological processes.

Full text of DOI:10.1016/j.bmc.2015.12.005

Bioorganic & Medicinal Chemistry 24 (2016) 220–225
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Recombinant thermoactive phosphoenolpyruvate carboxylase
(PEPC) from Thermosynechococcus elongatus and its coupling with
mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the
conversion of CO2 to oxaloacetate
Sonia Del Prete a, Viviana De Luca a, Clemente Capasso a, Claudiu T. Supuran b,c, Vincenzo Carginale a,
⇑
a Istituto di Bioscienze e Biorisorse—CNR, Via P. Castellino 111, 80131 Naples, Italy
b Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
c Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche, Polo Scientifico, Sesto Fiorentino, Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
With the continuous increase of atmospheric CO2 in the last decades, efficient methods for carbon cap-
ture, sequestration, and utilization are urgently required. The possibility of converting CO2 into useful
chemicals could be a good strategy to both decreasing the CO2 concentration and for achieving an effi-
cient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for
the catalytic conversion of CO2 mainly to bicarbonate have been implemented. In order to design and
construct a catalytic system for the conversion of CO2 to organic molecules, we implemented an
in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was con-
stituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobac-
terium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic
anhydrases (CAs), for converting CO2 into oxaloacetate, a compound of potential utility in industrial pro-
cesses. The catalytic procedure is in two steps: the conversion of CO2 into bicarbonate by CA, followed by
the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of
Received 15 October 2015
Revised 30 November 2015
Accepted 4 December 2015
Available online 8 December 2015
Keywords:
Phosphoenolpyruvate carboxylase
Carbonic anhydrase
CO2 conversion
Thermostability
Oxaloacetate
oxaloacetate (OAA). All tested CAs, belonging to a-, b-, and c-CA classes, were able to increase OAA pro-
duction compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested
in OAA production was in good agreement with the kinetic parameters for the CO2 hydration reaction of
these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the
extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was
achieved even if the two enzymes were exposed to temperatures up to 60 °C, suggesting a possible role
of the two coupled enzymes in biotechnological processes.
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
to the formation of only one molecule each of 3PGA and 2-phos-
phoglycolate (2PG), thus reducing the carbon fixation rate (Fig. 1A).
All plants depend particularly on the enzyme ribulose-1,5-bis-
phosphate carboxylase/oxygenase (RubisCO), which enables them
to fix all needed cellular carbon from the atmospheric gas CO2.
However, this enzyme is unable to discriminate between CO2 and
O2, leading to an oxygenase side reaction due to the binding of oxy-
gen in the CO2 binding site of RubisCO.1–3 Indeed, the oxygenation
of the substrate ribulose-1,5-bisphosphate (RuBP) results in
photorespiration, which instead of producing two molecules of
3-phosphoglycerate (3PGA) by the carboxylation reaction, leads
To minimize the loss of fixed carbon by photorespiration,
cyanobacteria and plants have evolved strategies leading to the
production of the enzyme phosphoenolpyruvate carboxylase
(PEPC).4,5 This enzyme is as an efficient CO2 fixating molecule that
catalyzes the irreversible b-carboxylation of phosphoenolpyruvate
(PEP) to form oxaloacetate (OAA) and inorganic phosphate, with
Mg2+ and Mn2+ as essential cofactors (Fig. 1B). PEPC mediates the
initial fixation of atmospheric carbon in light conditions, during
C4 and CAM photosynthesis, being widespread in plants, green
algae, and some types of bacteria, including cyanobacteria. PEPC
is absent in any animals and fungi. It has a high affinity for HCO3ꢀ
and is not inhibited by O2, it supplies carbon for the production
⇑
Corresponding author. Tel.: +39 81 6132715.
E-mail address: vincenzo.carginale@cnr.it (V. Carginale).
http://dx.doi.org/10.1016/j.bmc.2015.12.005
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

S. Del Prete et al. / Bioorg. Med. Chem. 24 (2016) 220–225
221
Figure 1. Reactions catalyzed by RubisCO (A) and PEPC (B).
of cytochromes, chlorophyll a, phycobilins, and cyanophycin, and
plays an anaplerotic role by replenishing C4 dicarboxylic acids uti-
lized for both energy and biosynthetic metabolism.6–8
Another promising option among the various efforts attempted
to mitigate CO2 accumulation is the possibility to exploit catalytic
pathways for converting CO2 into value-added chemicals, materi-
als or fuels, in order to accomplish CO2 capture, sequestration
and utilization (CCU or CCSU). CCU has the potential to reduce both
CO2 emissions and depletion of fossil resources.11,17
Overwhelming evidence has shown that the rapid growth of
atmospheric CO2 concentration, consequent of the continuous
increase in the consumption and burning of fossil fuels, is a major
contributor to global warming and climate change with potentially
devastating effects on Earth’s life. CO2 capture and storage (CCS)
has been identified as the critical enabling technology to signifi-
cantly reduce CO2 emissions.9,10 Among the various methods that
have been exploited for catalyzing the CO2 fixation/conversion
reaction, the enzymatic method, which is inspired by the CO2
metabolic process in cells, may provide an eco-friendly and
promising way for its superior stereo-specificity and region/
chemo-selectivity.11
Carbonic anhydrases (CAs, E.C. 4.2.1.1) are among the fastest
known enzymes, catalyzing the hydration of CO2 into bicarbonate
and a proton at turnover numbers approaching 106 sꢀ1. These
enzymes are practically ubiquitous in nature (mammals, plants,
algae, bacteria, and archaea), and represent an archetypal example
Most of the carbon fixed during light is converted into OAA,
which undergoes reduction to form different C4 acids, such as
aspartate and malate, as products of PEPC activity. Therefore, the
C4 acids synthesized by PEPC could be used to replenish the TCA
cycle intermediates, which in turn are used for biosynthetic reac-
tions. Moreover, in the field of industry, C4 dicarboxylic acids are
considered building-block chemicals with wide applications and
huge markets. These chemicals are commonly obtained from pet-
roleum, which is an unsustainable resource because of greenhouse
gas emissions produced from its use.18 It is obvious that petro-
leum-based chemical production must be shifted to biotechnolog-
ical processes. Recently, Chang et al. realized a relatively simple
multienzyme in vitro system to produce oxaloacetate from PEP
and CO2.19 In this system, the catalytic process mainly consisted
of two single-enzyme routes: the hydration of CO2 into bicarbonate
by CA, followed by the carboxylation with bicarbonate of PEP to
form OAA, catalyzed by PEPC.
of convergent evolution, comprising at least six distinct classes (
b, , d, f, and ) all requiring a divalent metal ion for catalysis to
take place. The -, b-, and d-classes contain Zn(II), -CAs are Fe
a,
c
g
a
c
(II) enzymes (but they are active also with bound Zn(II) or Co(II)
In the present paper we cloned and produced the recombinant
PEPC from the thermophilic cyanobacterium Thermosynechococcus
elongatus in order to construct an in vitro OAA production system
ions), the metal ion is usually replaced by cadmium in the f-CAs,
while
g
-CAs probably require Zn(II).12–14 Recently, carbonic anhy-
drases, owing to their extremely high catalytic efficiency, have
been identified as having great potential to accelerate CO2 capture
from large combustion emitters.15,16
stable at high temperature. For this reason, recombinant
-CA classes identified in the genome of mesophilic and ther-
mophilic bacteria have been coupled with this thermoactive PEPC.
a-, b-, and
c

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S. Del Prete et al. / Bioorg. Med. Chem. 24 (2016) 220–225
100 °C.22,37–41 The recombinant CAs were overexpressed and iso-
2. Results and discussion
lated from Escherichia coli (DE3) cells extract. After sonication
and centrifugation, the carbonic anhydrase activity was recovered
in the soluble fraction of cell extract. Using the affinity column
(His-select HF Nickel affinity gel), the aforementioned CAs were
purified to apparent homogeneity, as indicated by SDS–PAGE (data
not shown).
OAA and its derivatives have great potential in the organic acid
industry owing to their broad applications in the food industry as
additives and flavoring agents, and in pharmaceuticals as supple-
ments. Several reports provide evidence on the potential applica-
bility of OAA as a novel and powerful neuroprotective agent for
the treatment of ischemic stroke patients. The neuroprotective
effect of OAA is based in its capacity to induce a reduction of blood
glutamate as a result of the activation of the blood-resident
enzyme glutamate–oxaloacetate transaminase.25–27 In addition, it
has been reported that when OAA is administered to mice, it pro-
motes brain mitochondrial biogenesis, activates the insulin-signal-
ing pathway, reduces neuroinflammation, and activates
hippocampal neurogenesis.28
2.2. Kinetic parameters of TelPEPC and bacterial CAs
Kinetic parameters of the recombinant TelPEPC were deter-
mined at room temperature at pH 8.5 as described in the Section 4.
Vmax value was 22.5 (unit: mgꢀ1), whereas Km value for PEP was
0.55 mM.
Using the stopped-flow technique and CO2 as substrate, the
kinetic parameters were determined for the purified recombinant
bacterial CAs. The CO2 hydration reaction was monitored with
2.1. Preparation of TelPEPC and bacterial CAs
and without a catalyst at pH 7.5 for
b-CAs. In Table 1 is showed a comparison of the kinetic parameters
of the two human -CA isoenzymes (hCA I and hCA II) with those
of the VchCA, VchCAb, PgiCA, and the thermostable SazCA. It may
be observed that the mesophilic bacterial enzymes (VchCA,
VchCAb, and PgiCA) showed kinetic parameters similar to those
obtained for the human isoenzyme hCA I, while the thermostable
and thermoactive SazCA showed an activity level higher than the
a-CAs and at pH 8.3 for the
In order to obtain an OAA production system active at 25 °C as
well as higher temperatures, we first cloned and expressed in
Escherichia coli the PEPC gene (Tel-PEPC) from the cyanobacterium
T. elongatus, a microorganism able to grow in hot springs at tem-
peratures of about 55 °C. Thermophiles, owing to the temperature
range at which they live, are considered a good source of ther-
mostable enzymes, which are more tolerant not only to high tem-
peratures, but also to various physico-chemical conditions,
compared to their mesophilic counterparts.29,30 The recombinant
TelPEPC was overexpressed and isolated from Arctic Express DE3
cells extract. Most of PEPC activity was recovered from soluble
fraction of the cell extract after sonication and centrifugation, as
described in the Section 4. TelPEPC was purified to apparent homo-
geneity by affinity chromatography, as indicated by a single band
of about 110 kDa on SDS–PAGE (Fig. 2).
a
human a-CA or bacterial isoforms considered here (Table 1).
2.3. Conversion of CO2 to OAA by TelPEPC and bacterial CAs
In Figure 3 is shown the time-course of OAA production. This
experiment was performed in the presence of 5 mU of Tel-PEPC
with and without 30 WAU (Wilbur–Anderson units) of VchCA
(enzyme belonging to the a-CA class). OAA production was quan-
tified by using a colorimetric assay kit (see the Section 4). We
In combination with TelPEPC, we tested the following bacterial
CAs, obtained in recombinant form from our laboratory: the
from Vibrio cholerae, VchCA;20,31–33 the ß-CA from V. cholerae,
VchCAß; the
-CA from Porphyromonas gingivalis, PgiCA;21,34–36
and the extremophilic -CA from Sulphurhydrogenibium azorense,
a-CA
c
a
SazCA, which is able to retain its activity at temperatures up to
Figure 3. Time dependence of OAA production in the presence of 5 mU TelPEPC
alone or in combination with 30 WAU of VchCA. Assay conditions are described in
the Section 4. The values are the mean of three independent experiments SD.
Figure 2. Coomassie-stained SDS–PAGE of purified recombinant TelPEPC. Lane 1,
molecular mass markers. Lane 2, TelPEPC (1 lg).
Table 1
Kinetic parameters* for the CO2 hydration reaction catalyzed by the human cytosolic isozymes hCA I and II, VchCA, VchCAb, SazCA, and PgiCA
Isozyme
Organism
Activity level
Class
kcat (sꢀ1
)
Kcat/Km (Mꢀ1 ꢁ sꢀ1
)
hCA I
Homo sapiens
Homo sapiens
Vibrio cholerae
Sulfurihydrogenibium azorense
Vibrio cholerae
Moderate
Very high
Moderate
Highest
Moderate
Moderate
a
a
a
a
b
2.00 ꢁ 105
1.40 ꢁ 106
8.23 ꢁ 105
4.40 ꢁ 106
3.34 ꢁ 105
4.1 ꢁ 105
5.0 ꢁ 107
1.5 ꢁ 108
7.0 ꢁ 107
3.5 ꢁ 108
4.1 ꢁ 107
5.4 ꢁ 107
hCA II
VchCA
SazCA
VchCAb
PgiCA
Porphyromonas gingivalis
c
*
For each measurement at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity (see Experimental section).

S. Del Prete et al. / Bioorg. Med. Chem. 24 (2016) 220–225
223
noticed that the highest OAA titer was reached at 30 min of incu-
bation, either in presence or absence of VchCA. The VchCA-cat-
alyzed reaction induced a stimulatory effect on OAA production,
increasing the conversion rate of CO2 to bicarbonate. The maxi-
mum stimulatory effect was reached after 10 min, with a +2.6 fold
increase in OAA titer, see Figure 3.
In Figure 4 is reported the variation of OAA production as a
function of the amount of bacterial CAs (VchCA, panel A; VchCAß,
panel B; PgiCA, panel C). The assays were performed with an
incubation time of 30 min. From these data, it appears evident that
all the three CAs were able to enhance OAA production. The most
efficient CA in stimulating OAA production was VchCA (OAA titer
ranged from +2.4 to +3.0 fold using 6 and 96 WAU, respectively)
followed by PgiCA (+1.5 fold for 20 WAU, and +1.7 fold for
80 WAU) and VchCAß (+1.4 fold for 80 WAU and +2.2 fold for
320 WAU). It is important to note that these data are in agreement
with the kinetic parameters of the three enzymes, which
demonstrate that the catalytic efficiency was in the order
VchCA > PgiCA > VchCAß. This is a strong support in favor of our
initial hypothesis, that an effective CA enzyme will increase the
amount of OAA produced by the PEPC activity.
2.4. OAA production at high temperatures
In order to evaluate the OAA production system at higher tem-
peratures, the OAA assay was performed by coupling the ther-
mostable TelPEPC and the extremophilic SazCA. The commercial
PEPC from Sigma–Aldrich (PEPC Sigma) was also tested as a com-
parison with a mesophilic enzyme. In Figure 5 is shown the ther-
mal inactivation of TelPEPC, PEPC Sigma, and SazCA. All the
enzymes were incubated for 15 min at the temperatures indicated
on the x-axis. It can be noticed that there was no loss in activity
when TelPEPC was incubated at 40 °C, whereas it was observed a
slight reduction in OAA production at 50 °C and 60 °C. At these
temperatures, the TelPEPC residual activity was of 84% and 71%,
respectively, while at 70 °C the T. elongatus enzyme was com-
pletely inactivated. In contrast, PEPC Sigma was quickly inactivated
at 40 °C (34% of residual activity) and its activity was completely
lost after incubation at 50 °C (Fig. 5A). Moreover, coupling TelPEPC
and SazCA exposed at temperatures above the room temperature
was observed to be synergistic on OAA production, and this was
not affected by temperatures up to 60 °C (Fig. 5B). Our data thus
demonstrate that TelPEPC is resistant to higher temperatures com-
pared to the commercial and mesophilic PEPC Sigma and, when
used in combination with a thermostable CA, the system was not
inactivated even if the two enzymes were heated at temperatures
up to 60 °C.
Figure 5. Effect of temperature on OAA production. (A) Thermal inactivation of
TelPEPC (5 mU) and PEPC Sigma (5 mU). Both enzymes were incubated at the
indicated temperature for 15 min, cooled on ice, and then assayed as described in
Section 4. The activity at room temperature was set at 100%. (B) Thermal
inactivation of TelPEPC with and without 50 WAU of SazCA. Heat inactivation and
assay conditions were as described above. The values are the mean of three
independent experiments SD.
Figure 4. Effect of increasing amounts of bacterial CAs on OAA production (VchCA,
panel A; VchCAß, panel B; PgiCA, panel C). Assays were performed in presence of
5 mU of TelPEPC with an incubation time of 30 min. See Section 4 for further details.
The values are the mean of three independent experiments SD.

224
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
at the link http://www.ncbi.nlm.nih.gov/protein using ‘phospho-
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.
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