Production and characterization of Escherichia coli glycerol dehydrogenase as a tool for glycerol recycling

Article abstract of DOI:10.1016/j.procbio.2013.01.011

NAD⁺-dependent glycerol (Gro) dehydrogenase (GroDHase) catalyzes the conversion of Gro into dihydroxyacetone (DHA), the first step for fermentative Gro metabolism in Escherichia coli. In this work, we cloned the gldA gene that codes for the E. coli GroDHase and homologously expressed, purified, and kinetically characterized the recombinant protein. To achieve this, the enzyme was over-produced using Gro supplemented growth medium and lactose as the inducer. The enzyme was highly purified using either pseudo-affinity chromatography or a simple heat-shock treatment, which is potentially valuable for industrial production of GroDHase. We detected efficient oxidation of Gro derived from biodiesel production to DHA by gas chromatography. The results presented in this work support recombinant GroDHase production in a biorefinery setting as a relevant tool for converting Gro into DHA for future biotechnological applications.

Full text of DOI:10.1016/j.procbio.2013.01.011

Process Biochemistry 48 (2013) 406–412
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Production and characterization of Escherichia coli glycerol dehydrogenase as a
tool for glycerol recycling
Claudia Vanesa Piattonia,1, Carlos María Figueroa , Matías Damián Asención Diez ,
a,1
a
a
b
b
a
Ivana Lorna Parcerisa , Sebastián Antu n˜ a , Raúl Alberto Comelli , Sergio Adrián Guerrero ,
a
a,∗
Alejandro José Beccaria , Alberto Álvaro Iglesias
a
Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Paraje El Pozo, S3000ZAA Santa Fe, Argentina
Instituto de Investigaciones en Catálisis y Petroquímica (UNL-CONICET), Santiago del Estero 2654, S3000AOJ Santa Fe, Argentina
b
a r t i c l e i n f o
a b s t r a c t
+
Article history:
NAD -dependent glycerol (Gro) dehydrogenase (GroDHase) catalyzes the conversion of Gro into dihy-
Received 28 December 2012
Accepted 24 January 2013
Available online 4 February 2013
droxyacetone (DHA), the first step for fermentative Gro metabolism in Escherichia coli. In this work, we
cloned the gldA gene that codes for the E. coli GroDHase and homologously expressed, purified, and
kinetically characterized the recombinant protein. To achieve this, the enzyme was over-produced using
Gro supplemented growth medium and lactose as the inducer. The enzyme was highly purified using
either pseudo-affinity chromatography or a simple heat-shock treatment, which is potentially valuable
for industrial production of GroDHase. We detected efficient oxidation of Gro derived from biodiesel
production to DHA by gas chromatography. The results presented in this work support recombinant
GroDHase production in a biorefinery setting as a relevant tool for converting Gro into DHA for future
biotechnological applications.
Keywords:
Biodiesel
Biorefinery
Biotechnology
Dihydroxyacetone
Glycerol
©
2013 Elsevier Ltd. All rights reserved.
1
. Introduction
to reach a superior electrocatalytic performance of E. coli cells [7].
More recently [8], overexpression of GroDHase and DHA kinase
in E. coli cells increased fermentative production of ethanol from
crude Gro. Despite these relevant applications, the kinetic and bio-
chemical properties of the purified recombinant enzyme have been
scarcely explored.
Glycerol (Gro) dehydrogenase (GroDHase, EC 1.1.1.6) is a widely
distributed enzyme amongst bacteria and fungi that belongs to
the medium-chain dehydrogenase/reductase (MDRase) superfam-
ily [1] and catalyzes the reversible NAD -dependent oxidation of
+
Gro into dihydroxyacetone (DHA) [2]. GroDHase is mainly involved
in Gro utilization as a carbon and energy source. In Escherichia coli,
the enzyme (EcoGroDHase) catalyzes the first step in fermentative
Gro metabolism to produce DHA. This product is then phosphory-
lated by a specific kinase (EC 2.7.1.29) and enters into the glycolytic
pathway [3]. EcoGroDHase is encoded by the gldA gene (1104 bp),
is a polypeptide of ∼40 kDa, and in solution it forms homodimeric
and homooctameric structures [4]. Nearly 60 years ago, Asnis and
Brodie [5] purified this enzyme directly from the bacterium using
a simple heat-shock protocol. In the last decade, the E. coli gldA
gene was recombinantly expressed in order to improve either the
capacity of redox cofactor regeneration in reverse micelles [6] or
Worldwide petroleum reserves are limited and petroleum-
derived fuels contribute to the greenhouse gas effect by increasing
atmospheric CO2 levels. Thus, it is imperative to find new pro-
cesses for fuel production from raw materials. Bioethanol and
biodiesel production is attractive because it is eco-friendly, its
products are biodegradable and renewable, and it gives us the
potential to manage the CO2 balance in the atmosphere [9,10].
Biodiesel, the alternative to petroleum-derived diesel, is produced
from vegetable oils or animal fats through trans-esterification with
ethanol or methanol (alcoholysis) [9]. This process generates ethyl
or methyl esters of fatty acids and ∼10% (v/v) of Gro as a major
byproduct [11]. As biodiesel production increased, Gro production
increased as well and currently exceeds its industrial demands. The
cost of Gro has decreased because it is being generated in huge
quantities from the biodiesel industry and it is not easily disposed
of due to its environmental toxicity. Nonetheless, Gro constitutes an
attractive and competitive carbon source because the reduced state
of its carbon atoms has potential for the conversion into other use-
ful chemicals such as 1,3-propanediol, DHA, ethanol, and organic
acids (like succinic, propionic, and citric acids) (Fig. 1) [12,13]. These
Abbreviations: DHA, dihydroxyacetone; Eco, Escherichia coli; FID, flame
ionization detector; GC, gas chromatography; Gro, glycerol; GroDHase, Gro dehy-
drogenase; IPTG, isopropyl-␤-d-thiogalactopyranoside.
∗ Corresponding author. Tel.: +54 342 457 5206x217; fax: +54 342 457 5206x217.
E-mail address: iglesias@fbcb.unl.edu.ar (A.Á. Iglesias).
These authors contributed equally to this work.
1
1
359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2013.01.011

C.V. Piattoni et al. / Process Biochemistry 48 (2013) 406–412
407
Fig. 1. Schematic diagram of the enzymes (EC number) and pathways involved in the biochemical transformation of Gro. EC 1.1.1.1, alcohol dehydrogenase; EC 1.1.1.6,
GroDHase; EC 1.1.1.202, 1,3-propanediol oxidorreductase; EC 1.2.1.10, acetaldehyde dehydrogenase; EC 1.2.7.1, pyruvate synthase; EC 2.3.3.1, citrate synthase; EC 2.7.1.29,
DHA kinase; EC 2.7.1.40, pyruvate kinase; EC 4.2.1.30, Gro dehydratase; Fdox and Fdred, oxidized and reduced ferredoxin, respectively. Dashed arrows mean that more than
one enzyme is involved in the conversion step.
compounds are the main precursors for economically valuable
products in organic synthesis, biocatalysis, and the cosmetics
industry [14]. This potential use of Gro in biotechnology establishes
the need for developing affordable tools that will improve and help
implement effective biorefinery recycling strategies [15].
(EcogldA) was cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and
E. coli TOP10 cells were transformed with this construct. The identity of the cloned
gene was confirmed by DNA sequencing (Macrogen, Seoul, Korea). The EcogldA gene
was subcloned into the pET22b (Merck, Darmstadt, Germany) plasmid, obtaining
the construct [pET22b/EcogldA] that expresses a recombinant EcoGroDHase with a
6
His -tag at the C-term with no addition of extra amino acid.
Recently, several authors highlighted the potential of enzymes
to be used as efficient, economical, and environmentally friendly
biocatalysts [16,17], particularly in the biodiesel industry [18].
For instance, Luna et al. [19] successfully used covalently immo-
bilized pig pancreatic lipase for obtaining a second generation
biodiesel. In this context, we report the molecular cloning and
homologous recombinant over-expression of the gene encoding for
EcoGroDHase. We optimized the production of a highly pure state
of the enzyme and determined its kinetic properties and capacity
for using Gro derived from the biodiesel industry. Results are ana-
lyzed in the framework of the importance of the potential use of
the enzyme in the biorefinery processes to convert the byproduct
Gro into more valuable chemicals, primarily DHA.
2.3. Protein expression, culture conditions and media
Unless otherwise stated, E. coli BL21 (DE3) cells transformed with
◦
[
pET22b/EcogldA] were grown at 30 C with shaking at 200 rpm in LB medium
supplemented with 100 ␮g/ml ampicillin. Over-expression of the recombinant
enzyme was induced at OD600 of 0.4–0.6 with 0.5 mM IPTG (isopropyl-␤-D-thio-
galactopyranoside) or 2.5 g/l lactose for 5 h. Cell cultures were centrifuged at
◦
5
000 × g for 10 min and cells were stored at −20 C until use. For optimization of
recombinant GroDHase expression, several media were assayed under different
concentrations of lactose, temperature and induction time. The different media
used were: LB (5 g/l yeast extract, 10 g/l peptone, 10 g/l NaCl); GB (30 g/l yeast
extract, 27.5 g/l peptone, 1 g/l NaCl, 1.4 g/l Gro); and A1 (43 g/l yeast extract, 20 g/l
peptone, 18 g/l NaCl, 0.3 g/l Gro).
2
.4. Enzyme purification
2. Materials and methods
Harvested cells were resuspended in Buffer A (25 mM Tris–HCl pH 7.5, 300 mM
NaCl, 10 mM imidazole), disrupted by sonication and centrifuged to separate soluble
and insoluble fractions. The soluble sample was loaded onto a metal affinity resin
column (HisTrap, GE Healthcare Life Sciences, Piscataway, NJ, USA) charged with Ni2+
and pre-equilibrated with Buffer A. After extensive washing, the column was eluted
with a linear gradient of 10 to 300 mM imidazole in Buffer A. As an alternative to col-
umn chromatography, the recombinant EcoGroDHase was purified by heat-shock
2
.1. Bacterial strains and chemicals
E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA) were used for cloning pro-
cedures and plasmid maintenance. E. coli BL21 (DE3) cells (Invitrogen) were used
for protein expression. Enzymes used for molecular biology procedures were from
Fermentas (Glen Burnie, MD, USA). Gro and 1-butanol standards were acquired
from Merck (Darmstadt, Germany) and BDH Laboratory Reagents (Poole Dorset,
UK), respectively. DHA and NAD(H) were from Sigma (St. Louis, MO, USA). All the
other chemicals were of the highest quality available.
◦
treatment. Briefly, the soluble fraction was heated for 10 min at 60 C followed by
incubation on an ice-cold bath [5]. After 30 min, precipitated proteins were sepa-
◦
rated by centrifuging 10 min at 4 C and 30,000 × g. Active fractions were pooled,
◦
fractionated and stored at −80 C until use. Under these conditions the enzyme
remained fully active for at least three months.
2.2. Molecular cloning and subcloning of the E. coli gldA gene
The gldA gene was amplified by PCR from genomic DNA of the E. coli JM109
2.5. Protein methods
strain (New England BioLabs, Ipswich, MA, USA) using the specific oligonucleotides:
ꢀ
ꢀ
ꢀ
5
5
-CATATGGACCGCATTATTCAATCACCAGG-3 (forward, NdeI site is underlined) and
Protein concentration was determined by the Bradford method [20], with BSA
as a standard. Protein electrophoresis under denaturing conditions (SDS-PAGE) was
carried out on discontinuous 12% polyacrylamide gels as described by Laemmli [21].
Molecular mass standards for SDS-PAGE (GE Healthcare Life Sciences) included
ꢀ
-CTCGAGTTCCCACTCTTGCAGGAAACGCTG-3 (reverse, XhoI site is underlined).
◦
◦
◦
Amplification conditions were: 95 C for 5 min; 29 cycles at 94 C 1 min, 55 C for
9 s, and 72 C for 2 min; and a final step of 72 C for 10 min. The amplified gene
◦
◦
4

4
08
C.V. Piattoni et al. / Process Biochemistry 48 (2013) 406–412
phosphorylase b (97 kDa), BSA (66 kDa), ovalbumin (45 kDa), and carbonic anhy-
drase (30 kDa).
medium composition, inducer, induction conditions, pH, time, and
temperature) plays an important role in enzyme production [23].
We considered various alternative expression procedures to max-
imize the yield of the His-tagged EcoGroDHase using E. coli BL21
(DE3) cells transformed with the [pET22b/EcogldA] construct. First,
we evaluated GB and A1 as alternative culture media to standard
LB (Fig. 2A), both containing higher amounts of nutrients (yeast
extract and peptone), supplemented with Gro and with differ-
ent NaCl concentrations (see Section 2 for further details). The
A1 medium has advantages over LB and has been used previously
in our laboratory for producing other recombinant proteins [24].
However, A1 was not optimal for the expression of EcoGroDHase,
as it rendered a 4-fold lower yield than LB (Fig. 2A). Conversely, the
use of GB medium provided the highest recovery of recombinant
EcoGroDHase in crude extracts, with values nearly 3- and 10-fold
higher than LB and A1 media, respectively (Fig. 2A).
2.6. Native molecular mass determination
To determine the molecular mass of the native recombinant protein, the purified
enzyme was subjected to gel filtration chromatography. Typically, 50 ␮l of sample
were loaded onto a 4 ml Superdex 200 HR resin contained in a Tricorn 5/200 col-
umn (GE Healthcare Life Sciences) previously equilibrated with Buffer G (25 mM
Tris–HCl pH 8.0, 100 mM NaCl). The molecular mass was calculated using the cal-
ibration curve constructed with standard proteins (GE Healthcare Life Sciences),
including thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbu-
min (75 kDa) and ovalbumin (44 kDa). The column void volume was determined
using Blue Dextran loading solution (Promega).
2.7. Activity assay
GroDHase activity was assayed as described previously [5], with minor modifi-
cations. In the direction of Gro oxidation, the standard medium contained 100 mM
+
CAPS-NaOH pH 10.5, 5 mM NAD , and 500 mM Gro. In the direction of DHA reduc-
Based on these results, we selected GB as an effective cul-
ture medium for use with the E. coli BL21 (DE3) [pET22b/EcogldA]
expression system. To further improve the conditions of protein
expression, we evaluated the use of an alternative inducer as well
as time and temperature conditions after induction (Fig. 2B–D). For
instance, replacement of IPTG by lactose is a valuable alternative for
industrial protein production because it is an economical substitute
for induction of recombinant proteins. We found that IPTG could
be replaced by lactose for successful expression of EcoGroDHase,
with a maximum over-expression at 20 g/l of the disaccharide
(Fig. 2B). Interestingly, the recombinantenzymewas similarlyover-
produced when protein expression was triggered by addition of
tion, the standard medium was composed of 100 mM MES-NaOH pH 6.5, 0.2 mM
NADH, and 200 mM DHA. Alternatively, 50 mM phosphate/carbonate pH 10.3 (Gro
oxidation) and 6.5 (DHA reduction) were used as buffers. Both assays were initi-
ated by the addition of an appropriate amount of enzyme in a final volume of 50 ␮l.
◦
+
Reactions were performed at 30 C and the reduction/oxidation of NAD /NADH was
followed at 340 nm using a Multiskan Ascent 384-microplate reader (Thermo Elec-
tron Corporation, Waltham, MA, USA). One unit of enzyme activity (U) is defined
as the amount of enzyme catalyzing the formation of 1 ␮mol of product (NADH or
+
NAD ) in 1 min under the specified assay conditions.
2.8. Kinetic studies
Experimental data of enzyme activity were plotted against substrate con-
nH
nH
nH
nH
centration and fitted to the Hill equation: v= Vmax × S /(S0.5 + S ), using the
program Origin 7.0 (OriginLab Corporation). The term S0.5 is the concentration
of substrate (S) producing 50% of the maximal activity (Vmax), and nH is the Hill
coefficient. For activation studies, data were adjusted to a modified Hill equation:
0
.5 mM IPTG (Fig. 2C, lane 2) or 2.5 g/l lactose (Fig. 2C, lane 3).
Thereafter, we analyzed EcoGroDHase expression as a function
of temperature and induction time (Fig. 2D). Following optimiza-
tion of media and induction conditions, and considering economic
effectiveness, we found that higher yields of EcoGroDHase can
be achieved with our recombinant expression system using GB
nH
nH
nH
nH
v= (Vmax − v0) × A /(A0.5 + A ), where v0 is the initial velocity, and A0.5 is the
concentration of activator (A) producing 50% of the maximal activation (Vmax).
Kinetic constants are means of at least two independent sets of data, which were
reproducible within ± 10%.
◦
medium and inducing with 2.5 g/l lactose at 37 C for 5 h. Compared
2.9. Enzyme stability and activity as a function of pH and temperature
to the initial conditions (LB medium and induction with 0.5 mM
◦
The behavior of EcoGroDHase was evaluated at different values of pH and tem-
IPTG at 30 C for 5 h), the yield of EcoGroDHase was increased 10-
◦
◦
perature. Data of enzyme activity measured from 4 C to 75 C were fitted to the
Arrhenius plot [22] to calculate the activation energy (Ea). Activity of the recombi-
nant protein in both directions of the reaction, Gro oxidation and DHA reduction,
was measured at pH values ranging from 3 to 12, using 50 mM phosphate/carbonate
fold.
Two different and highly effective protocols were developed
and compared for the purification of EcoGroDHase (Fig. 3). The
His-tagged enzyme was purified to near homogeneity through
immobilized metal affinity chromatography (IMAC) (Fig. 3, lane 2),
and almost the same purity was achieved using a simple heat-shock
treatment (Fig. 3, lane 3). Interestingly, the heat-treated enzyme
retained more than 90% of its catalytic capacity and reached a spe-
cific activity of 25 U/mg in the direction of Gro oxidation, a value
similar to that obtained with the IMAC purified enzyme. Fig. 3
also illustrates that the major protein band obtained after purifi-
cation of recombinant EcoGroDHase is a polypeptide of ∼40 kDa,
which is consistent with the mass of 39.8 kDa calculated from the
enzyme’s primary sequence. The purified enzyme was loaded onto
a Superdex 200 column and eluted as a protein of 320 kDa (data
not shown), indicating that the native structure of EcoGroDHase is
an octamer. This result is in good agreement with data reported
for GroDHases purified from their natural sources, including those
from E. coli [4], Cellulomonas spp. [25,26], and Schizosaccharomyces
pombe [27].
◦
adjusted to the desired pH. The stability of EcoGroDHase was evaluated at 30 C and
◦
6
0
C using 50 mM phosphate/carbonate pH 8.0 and 10.3. In both assays, aliquots
of the mixture were taken at regular time intervals and used to evaluate enzyme
activity in the direction of Gro oxidation using 50 mM phosphate/carbonate pH 10.3,
as previously described in Section 2.7.
2.10. Gas chromatography
Reactant and reaction products of EcoGroDHase were analyzed by gas chro-
matography (GC) using a Shimadzu GC-2014 chromatograph provided with a flame
ionization detector (FID), and a DB-20 column of 25 m length and 0.33 mm internal
diameter. Analyses were made using the following temperature program: 3 min at
◦
◦
◦
1
2
50 C, heating up to 210 C at 20 C/min, and maintaining this temperature during
5 min. Because some of the acids that can produce the chemical reaction of Gro oxi-
dation are not detected by FID, the internal standard method was used to quantify
Gro and DHA. Calibration curves for Gro and DHA were made using 1-butanol as the
standard. Then, samples were analyzed adding 1-butanol as the internal standard,
determining Gro conversion into DHA.
3
. Results and discussion
Our system and procedure developed for the production
and purification of the recombinant EcoGroDHase has significant
advantages for obtaining an enzyme to be used in industrial
processes. The high protein expression achieved with lactose is
particularly relevant, as the natural disaccharide is a byproduct of
milk industry. This constitutes a relatively inexpensive and non-
toxic inducer that is by far an economically convenient alternative
to IPTG [28]. In the same way, the possibility to employ heat-
treatment as a successful purification procedure makes it highly
3
.1. Expression and purification of EcoGroDHase
Although the E. coli gldA gene was successfully expressed as a
recombinant polypeptide in its homologous host to improve the
bioconversion capacity of bacterial cells [7,8], quantification and
purification of the recombinant enzyme, as well as a complete
characterization of its kinetic properties are still lacking. It has
been shown that optimization of physicochemical parameters (like

C.V. Piattoni et al. / Process Biochemistry 48 (2013) 406–412
409
Fig. 2. Optimization of recombinant EcoGroDHase expression in different culture media and induction conditions with lactose. (A) Yield [mg enzyme/g wet biomass] (Yp/x)
of recombinant GroDHase achieved in cultures of E. coli BL21 (DE3) [pET22b/EcogldA] with different culture media (LB, GB or A1) induced with IPTG 0.5 mM for 5 h. (B)
Yield of EcoGroDHase from cells grown in GB medium induced with increasing lactose concentrations. (C) Analysis of EcoGroDHase expression by 12% SDS-PAGE followed
by Coomassie Blue staining. Molecular mass markers (lane 1); soluble fraction from E. coli BL21 (DE3) [pET22b/EcogldA] after 5 h induction with 0.5 mM IPTG (lane 2); or
2
.5 g/l lactose (lane 3); soluble fraction from E. coli BL21 (DE3) [pET22b] after induction with 0.5 mM IPTG (lane 4). (D) Yield of EcoGroDHase as a function of induction time.
◦
◦
◦
Transformed cells were grown in GB medium, induced with 2.5 g/l lactose at 37 C (ꢀ), 30 C (᭹), 20 C (ꢁ), and harvested at different times.
feasible for large-scale production, practically simple and remark-
ably low-priced. In addition, increased enzyme production when
recombinant E. coli cells were grown in a culture medium supple-
mented with Gro reinforces the utility of the procedure.
3.2. Kinetic characterization and enzyme stability
Although EcoGroDHase has been previously purified [4,5], its
kinetic properties were poorly analyzed. It is highly relevant to
establish the most convenient pH and temperature conditions if
the intention is to use this enzyme in industrial processes. We first
studied the activity of the recombinant enzyme in a wide pH range
using phosphate/carbonate (Fig. 4A). As it has been reported for
the enzyme purified from E. coli [4] and other bacteria [26,27,29],
activity of recombinant EcoGroDHase in the direction of Gro oxida-
tion was higher at pH values between 10.0 and 11.0, whereas the
highest activity in the direction of DHA reduction was observed at
a pH ranging from 7.0 to 8.0 (Fig. 4A). It is clear from Fig. 4A that
at the respective optimum pH in one sense the opposite reaction is
more than one order of magnitude lower.
Next, we analyzed the influence of temperature on the activ-
ity and stability of EcoGroDHase (Fig. 4B and C). The activity of
EcoGroDHase in the direction of Gro oxidation showed a maxi-
◦
mum at 60 C and further temperature increases caused a loss of
enzyme activity (Fig. 4B). With this data we constructed the Arrhe-
nius plot (Fig. 4B, inset) to calculate the Ea for EcoGroDHase, which
was 50.3 kJ/mol. As previously shown [5], EcoGroDHase is relatively
◦
stable at 60 C, which was conveniently utilized for enzyme purifi-
cation. However, we found that thermal stability of the enzyme
is critically affected by the pH. Fig. 4C shows that EcoGroDHase
◦
was stable when incubated at 60 C and pH 8.0 and quickly inacti-
◦
vated at pH 10.3. Conversely, when incubated at 30 C the enzyme
Fig. 3. Purification of recombinant EcoGroDHase analyzed by 12% SDS-PAGE fol-
lowed by Coomassie Blue staining. Molecular mass markers (lane 1); EcoGroDHase
purified by IMAC (lane 2); soluble (lane 3); and insoluble (lane 4) fractions obtained
after heat-shock treatment of crude extracts containing recombinant EcoGroDHase.
was relatively stable at both pH values, losing only 20% and 40% of
activity after 4 h incubation at pH 8.0 or 10.3, respectively (Fig. 4C).
These results are critical for designing heat-shock treatment

4
10
C.V. Piattoni et al. / Process Biochemistry 48 (2013) 406–412
protein purification procedures and maintenance protocols, as well
as to establish conditions to measure enzyme activity.
It has been reported that GroDHases can be activated by mono-
valent cations such as NH4+ and K+ [4,26,27,29], a characteristic
that was also found for EcoGroDHase. Fig. 5 shows that in the Gro
oxidation direction the recombinant enzyme is activated nearly 4-
fold by NH Cl, whereas it is activated only 2-fold by KCl. In the DHA
4
Fig. 5. Activation of EcoGroDHase by monovalent cations. Activity of the recombi-
nant enzyme was measured with increasing concentrations of NH4Cl (circles) and
KCl (squares). Assays were performed in the direction of Gro oxidation (filled sym-
bols) or DHA reduction (open symbols) with CAPS-NaOH (pH 10.5) and MES-NaOH
(
pH 6.5), respectively.
reduction direction, almost 2-fold activation was observed for
either NH Cl or KCl. In the direction of Gro oxidation, the A
4
0.5
for NH Cl was 1.14 ± 0.08 mM (nH of 1.4), while in the direction
4
of DHA reduction the A0 was 0.67 ± 0.09 mM (nH of 1.0) (values
.5
determined from Fig. 5). When EcoGroDHase was assayed in the
presence of 30 mM NH Cl or 20 mM KCl, no significant changes
4
were observed in the profiles of activity/stability versus temper-
ature or pH (data not shown).
Subsequently, we determined the kinetic parameters for sub-
strates in both directions of the reaction at the respective pH values
for maximal activity, in the absence of effectors or in the presence
of 30 mM NH Cl or 20 mM KCl. Saturation curves for the substrates
4
of EcoGroDHase were fitted to the Hill equation (see Fig. S1), as
described under Section 2. Kinetic parameters for EcoGroDHase
reported in Table 1 are similar to those described for GroDHase
from Aerobacter aerogenes [29]. Noteworthy is the 4-fold increase
in Vmax produced by NH Cl in the direction of Gro oxidation (Fig. 5
4
and Table 1). This is a relatively inexpensive salt and its addition
to the reaction could efficiently increase the conversion of Gro into
DHA. Also striking is the 70-fold reduction in the S0.5 for DHA and
the 2-fold increase in the Vmax when 30 mM NH Cl was added to
4
the reaction mixture (Table 1). These results are in good agreement
with those reported for the enzyme purified from A. aerogenes [29].
The addition of 20 mM KCl produced a similar decrease in the S0.5
for DHA but the Vmax did not change significantly (Table 1).
Table 1
Kinetic parameters for EcoGroDHase obtained with commercial Gro. Data were cal-
culated in absence and presence of 30 mM NH4Cl or 20 mM KCl. Reactions were
performed in CAPS-NaOH pH 10.5 (Gro oxidation) and MES-NaOH pH 6.5 (DHA
reduction), as described under Section 2.
Substrate
Gro
Effector
S0.5 (mM)
Vmax (U/mg)
nH
None
NH4Cl
KCl
None
NH4Cl
KCl
None
NH4Cl
KCl
None
NH4Cl
KCl
76 ± 6
33 ± 1
142 ± 7
72 ± 5
33 ± 1
142 ± 7
72 ± 5
58 ± 4
125 ± 4
47 ± 2
58 ± 4
125 ± 4
47 ± 2
0.9 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
1.0 ± 0.1
0.7 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
0.9 ± 0.1
0.9 ± 0.1
1.8 ± 0.1
81 ± 24
47 ± 3
NAD+
DHA
0.81 ± 0.09
1.0 ± 0.1
3.2 ± 0.5
15 ± 3
Fig. 4. Analysis of EcoGroDHase activity and stability as a function of pH and tem-
perature. (A) Assays were performed in the direction of Gro oxidation (ꢀ) or DHA
reduction (᭹) in phosphate/carbonate adjusted to the desired pH. (B) Activity of
the recombinant enzyme was determined in the direction of Gro oxidation in phos-
phate/carbonate pH 10.3 at increasing temperatures. The inset shows the Arrhenius
0.22 ± 0.03
0.24 ± 0.02
0.05 ± 0.01
0.08 ± 0.01
0.07 ± 0.01
NADH
plot used to determine the Ea of EcoGroDHase. (C) The enzyme was incubated at 30
◦
(
circles) and 60 C (squares) in phosphate/carbonate pH 8.0 (filled symbols) or pH
1
0.3 (open symbols). Assays were performed as described under Section 2.

C.V. Piattoni et al. / Process Biochemistry 48 (2013) 406–412
411
Table 2
Kinetic parameters for EcoGroDHase obtained with Gro from biodiesel waste. Reac-
tions were performed in phosphate/carbonate buffer pH 10.3, in absence and
presence of 30 mM NH4Cl or 20 mM KCl, as described under Section 2.
Substrate
Gro
Effector
S0.5 (mM)
Vmax (U/mg)
nH
None
NH4Cl
KCl
None
NH4Cl
KCl
46 ± 6
13 ± 2
65 ± 4
111 ± 6
63 ± 3
65 ± 4
111 ± 6
63 ± 3
1.0 ± 0.1
1.1 ± 0.1
1.0 ± 0.1
1.0 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
24 ± 2
NAD+
0.8 ± 0.1
1.4 ± 0.2
1.1 ± 0.1
Fig. 6. Gro from biodiesel waste is efficiently used by recombinant EcoGroDHase.
Assays were performed in the absence (᭹) or presence (ꢀ) of 30 mM NH4Cl in
phosphate/carbonate pH 10.3, as described under Section 2.
It has been previously described that DHA inhibits Gro oxida-
tion [26,29,30]. Thus, we conducted product inhibition studies for
EcoGroDHase in the presence of 30 mM NH Cl. We found that the
4
inhibitory effect of DHA was more significant when analyzed with
+
variable concentrations of Gro rather than NAD . Curves of Gro with
varying concentrations of DHA at 5 mM NAD+ showed a competi-
tive inhibition pattern (Fig. S2). According to Cornish-Bowden [31],
a Kic of 0.31 mM was calculated for DHA from the secondary plot
app
app
of the slopes (Km
/Vmax
) versus DHA concentration (Fig. S2,
inset). This result is consistent with those reported for GroDHase
from A. aerogenes [29] and Candida valida [30], which were inhibited
Fig. 7. EcoGroDHase converts Gro into DHA. The product of the reaction catalyzed
by the recombinant enzyme was analyzed by GC, as described under Section 2. (A)
Chromatogram obtained for Gro from biodiesel waste. (B) Chromatogram obtained
for the product of the reaction catalyzed by EcoGroDHase using Gro from biodiesel
waste as a substrate. (C) The same chromatogram shown in (B) was magnified 100-
fold. (D) Control chromatogram obtained for a mixture of commercial DHA and
Gro.
by DHA. Although this is a relatively small K , it should be noted
ic
that the inhibition is competitive. If EcoGroDHase would be used in
a biorefinery there are at least two different ways to overcame DHA
inhibition: (i) Gro concentration should be always maintained high
enough and (ii) design a continuous system to remove the product
and maintaining it below an inhibitory level.
To determine if EcoGroDHase produced DHA by bioconver-
sion of Gro from biodiesel waste, we analyzed the product of the
reaction by GC. The enzymatic reaction was performed in phos-
phate/carbonate pH 10.3 with 0.78 M Gro from biodiesel waste
3.3. Kinetic characterization with biodiesel wasted Gro
(
commercial Gro was used as control in a separate reaction), 20 mM
To further explore the utility of EcoGroDHase for industrial
+
◦
NAD , and 0.4 mg/ml EcoGroDHase at 30 C. The reaction progress
was followed by measuring the absorbance of NADH at 340 nm
until it was constant. Next, the enzyme was removed by using a
Microcon-30 kDa centrifugal filter unit with Ultracel-30 membrane
applications, we evaluated the ability of the enzyme to use Gro from
biodiesel waste as substrate. The biodiesel waste was generated
by an industrial chemical process in a plant utilizing triglyc-
erides from Glycine max (producing 30,000 tons of biodiesel per
year). We determined the concentration of Gro in the biodiesel
waste solution by GC as 57.5% (w/v), which is equivalent to 7.8 M.
This solution also contained water (30%), methanol (2.5%), and
other organic compounds commonly found in biodiesel waste [32].
EcoGroDHase effectively used Gro from biodiesel waste as a sub-
strate (Fig. 6), and the curves of activity versus Gro concentration
were similar to those obtained with commercial Gro (data not
shown). We also determined the kinetic parameters of recombinant
EcoGroDHase using Gro from biodiesel waste. Table 2 shows that
in phosphate/carbonate buffer, which is considerably cheaper than
other commercially available buffers, the calculated parameters are
similar to those obtained using CAPS-NaOH and MES-NaOH (see
Table 1). Interestingly, a considerably higher Vmax was observed
with Gro from biodiesel waste when the activity was measured
without addition of effectors (Table 2). This behavior could be due
to the high ionic strength present in Gro from biodiesel waste,
which could contain a high concentration of KCl, commonly used
as a catalyst for transesterification of triglycerides with methanol
(Millipore, Billerica, MA, USA) and the filtrate was conveniently
diluted and assayed by GC. The product was DHA and the estimated
conversion of Gro into DHA was calculated as 2.2% (Fig. 7), which
is equivalent to 17 mM. This value was calculated using calibra-
tion curves for Gro and DHA made using 1-butanol as the internal
standard. This quantity of DHA produced is reasonable because it
stoichiometrically agrees with the expected value since the ini-
+
tial concentration of the limiting reactant (NAD ) was 20 mM. We
should consider using coupled assays in the future to enzymatically
regenerate NAD+ since it is one of the most expensive reagents to
enzymatically convert Gro into DHA.
4. Conclusions
Production of biodiesel involves generation of relatively huge
amounts of Gro as a byproduct, which makes critical developing
tools for its recycling. Gro has a great potential to produce other
chemicals that could be used in different industrial processes. We
found that recombinant EcoGroDHase is a promising biocatalyst to
[
9].

4
12
C.V. Piattoni et al. / Process Biochemistry 48 (2013) 406–412
convert Gro from biodiesel waste into DHA as a biorefinery strategy.
In this work we report a thorough kinetic analysis of recombi-
nant EcoGroDHase. Our study includes convenient production of
the recombinant enzyme at a relatively low cost. The growth of
transformed cells producing EcoGroDHase was efficiently achieved
with a medium containing Gro, thus representing an extra use of the
byproduct. Also, induction of recombinant protein expression was
optimized with lactose, an inexpensive chemical. Development of
biorefineries is a key step to reach integrated production of bio-
fuels, chemicals, and materials, making the process sustainable,
environment-friendly and also economically more feasible.
[10] Yang F, Hanna MA, Sun R. Value-added uses for crude glycerol – a byproduct of
biodiesel production. Biotechnol Biofuels 2012;5:13.
[
11] Ma F, Hanna MA. Biodiesel production:
999;70:1–15.
a review. Bioresour Technol
1
[12] da Silva GP, Mack M, Contiero J. Glycerol: a promising and abundant carbon
source for industrial microbiology. Biotechnol Adv 2009;27:30–9.
[
13] Almeida JR, Favaro LC, Quirino BF. Biodiesel biorefinery: opportunities and chal-
lenges for microbial production of fuels and chemicals from glycerol waste.
Biotechnol Biofuels 2012;5:48.
[14] Corma A, Iborra S, Velty A. Chemical routes for the transformation of biomass
into chemicals. Chem Rev 2007;107:2411–502.
[
[
[
[
[
[
15] Posada JA, Rincon LE, Cardona CA. Design and analysis of biorefineries
based on raw glycerol: addressing the glycerol problem. Bioresour Technol
2012;111:282–93.
16] Savile CK, Janey JM, Mundorff EC, Moore JC, Tam S, Jarvis WR, et al. Biocat-
alytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin
manufacture. Science 2010;329:305–9.
17] Siegel JB, Zanghellini A, Lovick HM, Kiss G, Lambert AR, St Clair JL, et al.
Computational design of an enzyme catalyst for a stereoselective bimolecular
Diels–Alder reaction. Science 2010;329:309–13.
18] Hama S, Konda A. Enzymatic biodiesel production: an overview of
potential feedstocks and process development. Bioresour Technol 2012
http://dx.doi.org/10.1016/j.biortech.2012.08.014
19] Luna D, Posadillo A, Caballero V, Verdugo C, Bautista FM, Romero AA, et al. New
biofuel integrating glycerol into its composition through the use of covalent
immobilized pig pancreatic lipase. Int J Mol Sci 2012;13:10091–112.
20] Bradford MM. A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding. Anal
Biochem 1976;72:248–54.
Acknowledgements
The authors want to thank Dr. Misty L. Kuhn for critically reading
the manuscript. This work was supported by grants from ANPCyT
(
PICT’07 668 and PICT’08 1754), UNL (CAI+D Redes and Orientado),
and CONICET (PIP 2519). MDAD is a CONICET Doctoral Fellow. CVP,
CMF, RAC, SAG, and AAI are Researchers from the same institution.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.procbio.2013.01.011.
[21] Laemmli UK. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 1970;227:680–5.
[
22] Segel IH. Enzyme kinetics: behavior and analysis of rapid equilibrium and
steady state enzyme systems. New York: John Wiley & Sons; 1993.
[
23] Liu JF, Zhang ZJ, Li AT, Pan J, Xu JH. Significantly enhanced production of recom-
binant nitrilase by optimization of culture conditions and glycerol feeding. Appl
Microbiol Biotechnol 2010;89:665–72.
References
[
24] Giordano PC, Martinez HD, Iglesias AA, Beccaria AJ, Goicoechea HC. Application
of response surface methodology and artificial neural networks for optimiza-
tion of recombinant Oryza sativa non-symbiotic hemoglobin 1 production by
Escherichia coli in medium containing byproduct glycerol. Bioresour Technol
[
[
[
[
1] Persson B, Hedlund J, Jornvall H. Medium- and short-chain dehydroge-
nase/reductase gene and protein families: the MDR superfamily. Cell Mol Life
Sci 2008;65:3879–94.
2] Ruzheinikov SN, Burke J, Sedelnikova S, Baker PJ, Taylor R, Bullough PA, et al.
Glycerol dehydrogenase: structure, specificity, and mechanism of a family III
polyol dehydrogenase. Structure 2001;9:789–802.
3] Durnin G, Clomburg J, Yeates Z, Alvarez PJ, Zygourakis K, Campbell P, et al.
Understanding and harnessing the microaerobic metabolism of glycerol in
Escherichia coli. Biotechnol Bioeng 2009;103:148–61.
4] Tang CT, Ruch Jr FE, Lin CC. Purification and properties of a nicotinamide ade-
nine dinucleotide-linked dehydrogenase that serves an Escherichia coli mutant
for glycerol catabolism. J Bacteriol 1979;140:182–7.
2010;101:7537–44.
[
[
[
25] Leichus BN, Blanchard JS. Isotopic analysis of the reaction catalyzed by glycerol
dehydrogenase. Biochemistry 1994;33:14642–9.
26] Nishise H, Nagao A, Tani Y, Yamada H. Further characterization of glycerol dehy-
drogenase from Cellulomonas sp. NT3060. Agric Biol Chem 1984;48:1603–9.
27] Marshall JH, May JW, Sloan J. Purification and properties of glycerol: NAD+ 2-
oxidoreductase (glycerol dehydrogenase) from Schizosaccharomyces pombe. J
Gen Microbiol 1985;131:1581–8.
[
28] Menzella HG, Ceccarelli EA, Gramajo HC. Novel Escherichia coli strain allows
efficient recombinant protein production using lactose as inducer. Biotechnol
Bioeng 2003;82:809–17.
[5] Asnis RE, Brodie AF. A glycerol dehydrogenase from Escherichia coli. J Biol Chem
1
953;203:153–9.
[
6] Ichinose H, Kamiya N, Goto M. Enzymatic redox cofactor regeneration in organic
media: functionalization and application of glycerol dehydrogenase and solu-
ble transhydrogenase in reverse micelles. Biotechnol Prog 2005;21:1192–7.
7] Xiang K, Qiao Y, Ching CB, Li CM. GldA overexpressing-engineered E. coli
as superior electrocatalyst for microbial fuel cells. Electrochem Commun
[
29] McGregor WG, Phillips J, Suelter CH. Purification and kinetic characterization of
a monovalent cation-activated glycerol dehydrogenase from Aerobacter aero-
genes. J Biol Chem 1974;249:3132–9.
30] Gärtner G, Kopperschläger G. Purification and properties of glycerol dehydro-
genase from Candida valida. J Gen Microbiol 1984;130:3225–33.
31] Cornish-Bowden A. Fundamentals of Enzyme Kinetics. London: Portland Press;
[
[
[
[
[
[
2
009;11:1593–5.
8] Cintolesi A, Clomburg JM, Rigou V, Zygourakis K, Gonzalez R. Quantitative anal-
ysis of the fermentative metabolism of glycerol in Escherichia coli. Biotechnol
Bioeng 2011;109:187–98.
2002.
32] Chatzifragkou A, Papanikolaou S. Effect of impurities in biodiesel-derived waste
glycerol on the performance and feasibility of biotechnological processes. Appl
Microbiol Biotechnol 2012;95:13–27.
9] Vasudevan PT, Briggs M. Biodiesel production – current state of the art and
challenges. J Ind Microbiol Biotechnol 2008;35:421–30.