One-pot enzymatic reaction sequence for the syntheses of d-glyceraldehyde 3-phosphate and l-glycerol 3-phosphate

Article abstract of DOI:10.1016/j.molcatb.2015.12.004

A one-pot enzymatic reaction sequence for the synthesis of optically pure d-glyceraldehyde 3-phosphate (d-GAP) and l-glycerol 3-phosphate (sn-G3P) was designed using fructose-bisphosphate aldolase from rabbit muscle (RAMA), sn-glycerol 3-phosphate dehydrogenase (sn-G3PDH) and formate dehydrogenase from Candida boidinii (FDH). The reaction sequence significantly improves the aldol cleavage of d-fructose 1,6-bisphosphate (d-F16BP) catalyzed by RAMA and yields 100% conversion of d-F16BP by overcoming thermodynamic limitation. The degradation kinetics of d-GAP under reaction conditions was investigated and a reaction kinetics model defining the entire cascade was developed. Validation of the model shows 98.5% correlation between experimental data and numerically simulated data matrices. The evaluation of different types of reactor was performed by combining the reaction kinetics model, mass balances and kinetics of the non-enzymatic degradation of d-GAP. Batch-wise operation in a stirred tank reactor (STR) is the most convenient procedure for the one-pot enzymatic syntheses of d-GAP and sn-G3P. The separation of the two products d-GAP and sn-G3P has been achieved using polyethylenimine (PEI)-cellulose TLC.

Full text of DOI:10.1016/j.molcatb.2015.12.004

Journal of Molecular Catalysis B: Enzymatic 124 (2016) 77–82
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
One-pot enzymatic reaction sequence for the syntheses of
d-glyceraldehyde 3-phosphate and l-glycerol 3-phosphate
Getachew S. Mollaa, Roland Wohlgemuthb, Andreas Liesea,∗
a Institute of Technical Biocatalysis, Hamburg University of Technology, Denickestr. 15, D-21073 Hamburg, Germany
b Sigma–Aldrich Chemie GmbH, Industriestr. 25, Buchs, CH-9470 Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A one-pot enzymatic reaction sequence for the synthesis of optically pure d-glyceraldehyde 3-phosphate
(d-GAP) and l-glycerol 3-phosphate (sn-G3P) was designed using fructose-bisphosphate aldolase from
rabbit muscle (RAMA), sn-glycerol 3-phosphate dehydrogenase (sn-G3PDH) and formate dehydrogenase
from Candida boidinii (FDH). The reaction sequence significantly improves the aldol cleavage of d-fructose
1,6-bisphosphate (d-F16BP) catalyzed by RAMA and yields 100% conversion of d-F16BP by overcoming
thermodynamic limitation. The degradation kinetics of d-GAP under reaction conditions was investigated
and a reaction kinetics model defining the entire cascade was developed. Validation of the model shows
98.5% correlation between experimental data and numerically simulated data matrices. The evaluation
of different types of reactor was performed by combining the reaction kinetics model, mass balances and
kinetics of the non-enzymatic degradation of d-GAP. Batch-wise operation in a stirred tank reactor (STR) is
the most convenient procedure for the one-pot enzymatic syntheses of d-GAP and sn-G3P. The separation
of the two products d-GAP and sn-G3P has been achieved using polyethylenimine (PEI)-cellulose TLC.
© 2015 Published by Elsevier B.V.
Received 24 July 2015
Received in revised form
18 November 2015
Accepted 4 December 2015
Available online 8 December 2015
Keywords:
Phosphorylated metabolite
One-pot reaction sequence
Reaction kinetics
Reactor simulation
Downstream processing
1. Introduction
tailored with a desired fatty acid chain length and acyl-number as
well as position using sn-glycerol 3-phosphate O-acyltransferase
In vitro synthesis of optically pure metabolites is of much inter-
est in various applications such as enzyme substrates, biochemicals
for biomedical research, drug discovery and the discovery of new
metabolic pathways, standards for analytical technologies as well
as pharmaceuticals and food additives. Phosphorylated metabo-
lites have historically played a prominent role, are ubiquitous and
are therefore highly required for various applications. In synthetic
applications they are useful to design in vitro biocatalytic reaction
sequences using various aldolases or transketolases that allow syn-
thesizing novel products [1–12]. d-Glyceraldehyde 3-phosphate
(d-GAP) and l-glycerol 3-phosphate (sn-G3P) are essential phos-
phorylated metabolites occurring in various metabolic pathways,
e.g., d-GAP is a central metabolite in glycolysis, thiamine biosynthe-
sis [13], methylerythritol phosphate (MEP) pathway [14,15] and
photosynthetis [16,17]. sn-G3P is a key building block of phos-
pholipids in bacteria as well as eucarya and useful for an in vitro
preparation of optically pure ␣-glycerophospholipids that can be
[18–23]. The therapeutic effect of calcium glycerol phosphate has
recently been reported for preserving and/or treating of intestinal
integrity in ischemia [24,25].
Phosphorylation of glycerol by ATP catalyzed by glycerol kinase
(E.C. 2.7.1.30) has been reported as a major enzymatic synthesis
of sn-G3P [26,27]. As this reaction system requires stoichiomet-
ric amounts of the phosphoryl donor ATP, the scalability of this
reaction system for large-scale synthesis is limited. The scalability
of most reported ATP regeneration systems, which require more
expensive co-substrates like phosphoenolpyruvate [1,26,28] and
acetyl phosphate [1,27,29], is even more limited. Several multistep
chemical synthetic routes have been reported for the preparation of
d-GAP, e.g., a 9 step sequence starting from a kilogram of d-mannitol
to produce few grams of d-GAP using toxic reagents like HgCl2 and
HgO [30], a 10 step sequence starting from 2-O-benzyl-d-arabinose
[31], oxidation of d-fructose 6-phosphate (d-F6P) by Pb(OAc)4
[10,32], oxidative cleavage by H5IO6 of d-fructose 1,6-bisphosphate
(d-F16BP) [33] or of d-F6P [28]. Lack of selectivity makes purifi-
cation steps too laborious and drastically reduces product yields.
The reported enzymatic synthesis of d-GAP applying aldolase to
catalyze aldol cleavage of d-F16BP [34] has thermodynamics as
main drawback, being in favor of reverse aldol condensation with
an equilibrium constant of nearly 10−4 M [2,35]. To overcome this
∗
Corresponding author at: Institute of Technical Biocatalysis, Hamburg Uni-
versity of Technology (TUHH), Denickestr. 15, Hamburg D-21073, Germany.
Fax: +49 40428 78 2127.
E-mail address: liese@tuhh.de (A. Liese).
http://dx.doi.org/10.1016/j.molcatb.2015.12.004
1381-1177/© 2015 Published by Elsevier B.V.

78
G.S. Molla et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 77–82
D-GAP
RAMA
+
D-F16BP
sn-G3PDH
sn-G3P
DHAP
NAD+
HCOO-
NADH
FDH
CO2
Fig. 1. A one-pot cascade enzymatic reaction sequence for the syntheses of d-
GAP and sn-G3P; d-F16BP: d-fructose 1,6-bisphosphate, d-GAP: d-glyceraldehyde
3-phosphate, DHAP: dihydroxyacetone phosphate, RAMA: rabbit muscle aldolase,
sn-G3P: sn-glycerol 3-phosphate, sn-G3PDH: sn-glycerol 3-phosphate dehydroge-
nase and FDH: formate dehydrogenase.
Table 1
Fig. 2. Stability of NADH incubated in 50 mM TEA at 25 ◦C at different pH levels.
Activity and stability of RAMA, sn-G3PDH and FDH as a function of pH: 0.5 mM d-
F16BP, 0.5 mM NADH, 0.18 U/ml RAMA and 6.4 U/ml sn-G3PDH (for the activity assay
of RAMA); 0.5 mM DHAP, 0.5 mM NADH and 0.8 U/ml sn-G3PDH (for the activity
assay of sn-G3PDH) and 50 mM NaHCOO, 0.5 mM NAD+ and 6.5 U/ml FDH (for the
activity assay of FDH) in 50 mM TEA buffer at 25 ◦C. Incubation conditions: RAMA
(50 mM TEA buffer, 25 ◦C and 400 rpm); sn-G3PDH (50 mM TEA buffer, 25 ◦C and
400 rpm) and FDH (50 mM TEA buffer, 25 ◦C and 400 rpm).
and stability data of all the three enzymes involved in the reaction
sequence as a function of pH; moreover, detailed enzymes activity
and stability data are shown in the supplementary document. Con-
sidering that enzymes show maximum activity at different pH, the
activity loss of one enzyme at a pH where another enzyme shows
maximum activity and the different enzyme stabilities with respect
to pH, pH 8 appears to represent an optimum value.
Enzyme
RAMA
sn-G3PDH At pH 8
FDH At pH [7–9]
Maximum activity Loss of activity
Higher stability
At pH 6 11% at pH 7 and 35% at pH 8 At pH [7,8]
88% at pH 6 and 45% at pH 7 At pH [6–9]
0% at pH 8 At pH [6–8]
As shown in Fig. 2 and it has also been known that the reduced
form of the nucleotide cofactor (NADH) is unstable at acidic pH,
while it is stable at alkaline pH [36]. The oxidized form of the
nucleotide cofactor (NAD+) is on the other hand unstable at alkaline
pH, while it is stable at acidic pH [36]. The decrease of pH acceler-
ates NADH depletion, whereas the increase of pH accelerates NAD+
depletion. According to results of stability measurements for NADH
as well as for NAD+ [36], as function of pH, pH 8 appears to be
optimum for the stability of NADH and NAD+.
limitation by shifting the reaction equilibrium, the reaction was
carried out in the presence of hydrazine yielding d-GAP hydrazone
as final product instead of the desired d-GAP [34].
In consequence three main objectives of this work were defined,
starting with the design of a one-pot enzymatic reaction sequence
for the syntheses of d-GAP and sn-G3P without protection and
deprotection steps. The second objective was a comprehensive
reaction engineering characterization like activity, stability and
selectivity of all enzymes involved, stability of cofactors and
products, reaction kinetics model development and simulation
of different reactor types. To develop a downstream processing
(DSP) method was the third objective. In the synthesis of d-GAP,
fructose-bisphosphate aldolase from rabbit muscle (RAMA) (E.C.
4.1.2.13) catalyzed aldol cleavage of d-F16BP to d-GAP and dihy-
droxyacetone phosphate (DHAP) was used. In situ reduction of the
co-product DHAP to sn-G3P catalyzed by sn-glycerol 3-phosphate
dehydrogenase from rabbit muscle (sn-G3PDH) (E.C. 1.1.99.5) was
added as consecutive reaction step in order to shift the reaction
equilibrium and synthesize the second target product (sn-G3P).
Moreover, since the second reaction step requires the expensive
cofactor NADH, it was coupled with formate dehydrogenase from
Candida boidinii (FDH) (E.C. 1.2.1.2) catalyzing the in situ regenera-
tion of NADH. The entire reaction sequence is shown in Fig. 1.
The stabilities of triosephosphate metabolites such as DHAP and
d-GAP at neutral and alkaline pH conditions depend very much on
the molecular structure, e.g., whether the glyceraldehyde is phos-
phorylated in the 2- or 3-position, and the medium composition
[1,28]. A decomposition mechanism of eliminating the phosphate
group via an enediolate phosphate intermediate has been proposed
[37,38]. d-GAP is stable at pH of 4 or below while the enzymes
involved are not active and NADH shows a very low stability at this
pH region; therefore, the degradation kinetics of d-GAP at pH 8 is
a critical parameter for process optimization of the one-pot enzy-
matic reaction sequence. The degradation rate of d-GAP in 50 mM
TEA buffer at pH 8 and 25 ◦C can be defined by first order kinetics
with a rate constant of 2.3 × 10−5 s−1 and a corresponding half-life
time of 8.35 h. The degradation of d-GAP was investigated in dif-
ferent buffer media because catalysis of the phosphate elimination
reaction by a tertiary amine buffer and a rate increase with increas-
ing buffer concentration was described [37]. Our experiments show
the rates of d-GAP degradation in non-buffered aqueous medium,
100 mM TEA, 50 mM TEA and 100 mM potassium phosphate buffer
(PPB) media to be similar, while d-GAP stability is increased in
100 mM Tris–HCl buffer. Fig. 3 shows the degradation of d-GAP
incubated in different buffer media at pH 8. The stabilization of d-
GAP by Tris–HCl buffer may be due to interactions of d-GAP with the
Tris–HCl buffer [39]. Tris–HCl buffer is however not applicable as it
also affects the activity of sn-G3PDH due to interactions with DHAP
and additionally the use of inorganic phosphate buffer interferes
in product purification. Furthermore, the selectivity of sn-G3PDH
toward d-GAP and DHAP due to their structural similarity as well
as the NADH oxidase activity of all the three enzymes active in the
reaction sequence were examined. Results demonstrate 100% sn-
G3PDH selectivity toward to DHAP and no depletion of NADH in
the presence of all the three enzymes without d-F16BP addition.
2. Results and discussion
2.1. Reaction system optimization
The capability of the one-pot reaction sequence (shown in Fig. 1)
to shift the reaction equilibrium of RAMA catalyzed aldol cleavage
of d-F16BP was examined by performing reactions using excess
amount of NADH without coupling the cofactor regeneration sys-
tem. Results showed a significant improvement with reactions
yielding 100% conversions of d-F16BP. The selection of optimum
reaction conditions like pH is essential for developing an enzy-
matic process and a reaction kinetics model. Activities, stabilities
and selectivities of all three enzymes involved, stabilities of both
the reduced and oxidized nucleotide cofactor as well as stability of
d-GAP as a function of pH were investigated. Table 1 shows activity

G.S. Molla et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 77–82
79
Table 3
Kinetic properties and the values of kinetic constants for rabbit muscle aldolase
(RAMA), formate dehydrogenase (FDH) and sn-glycerol 3-phosphate dehydrogenase
(sn-G3PDH).
Kinetics parameters
RAMA
FDH
sn-G3PDH
Km,d-F16BP (mM)
Kic,d-GAP (mM)
Kiu,d-GAP (mM)
vmax (U/mg)
0.0065 0.002
0.11
0.40
6.80 0.2
−
Km,HCOO (mM)
6.08 2.2
+
Km,NAD (mM)
0.02 0.001
0.04 0.01
0.55 0.005
Kic,NADH (mM)
vmax (U/mg)
Km,DHAP (mM)
Km,NADH (mM)
vmax (U/mg)
0.049 [43]
0.28
0.0081 [44]
123
Fig. 3. Degradation of 20 mM d-glyceraldehyde 3-phosphate incubated in 50 mM
TEA buffer pH 8, 100 mM Tris–HCl buffer pH 8 or alternatively 100 mM potassium
phosphate buffer (PPB) pH 8 at 25 ◦C.
Table 2
The influence of co-substances present in the reaction mixture on the activity of rab-
bit muscle aldolase (RAMA), sn-glycerol 3-phosphate dehydrogenase (sn-G3PDH)
and formate dehydrogenase (FDH) involved in the reaction sequence.
Co-substance
RAMA
sn-G3PDH
FDH
d-F16BP
d-GAP
sn-G3P
NaHCOO
Native
Native
×
×
√
×
×
Native
×
√
×
Native
√
Indicates the co-substance suppresses the enzyme activity; ×indicates the co-
substance does not influence the enzyme activity.
Fig. 4. Graphical fitness of experimental data and numerically simulated data using
the kinetics models for batch reactions of RAMA catalyzed aldol cleavage of d-
F16BP and FDH catalyzed reduction of NAD+: 0.1 mM d-F16BP, 0.5 mM NADH,
0.0046 mg/ml RAMA, 0.044 mg/ml sn-G3PDH in 50 mM TEA pH 8 and 25 ◦C for
RAMA catalyzed aldol cleavage of d-F16BP and 0.0125 mM NAD+, 500 mM NaH-
COO, 0.075 mg/ml FDH in 50 mM TEA pH 8 and 25 ◦C for FDH catalyzed reduction of
NAD+.
2.2. Reaction kinetics model development
The investigation of biocatalytic reaction kinetics is essential for
identifying relevant kinetic parameters and to understand the reac-
tion mechanism. An appropriate reaction kinetics model is a major
tool and criterion to choose a specific reactor type and to optimize
the process [40–42]. Since the reaction sequence contains, in addi-
tion to the native substrate and product of each of the enzymes,
several compounds, their influence on each of the enzyme activities
was investigated. These activity data can be included in the overall
reaction kinetics model and are useful for selecting the concentra-
tion of each enzyme. Table 2 shows the influence on the activity of
enzymes involved. The dependence of the enzyme activities on the
concentrations of their native substrates and products has been
analyzed as well and taken into account in the development of
kinetic models.
The cascade reaction consists of three enzymes arranged in two
reversible consecutive reactions and the last one coupled with the
third irreversible cofactor regeneration. For the development of
the overall reaction kinetics model, this simplifies the entire reac-
tion sequence to a single substrate irreversible enzymatic reaction.
The kinetic behavior of each enzyme with respect to their native
substrate and product must be known in order to choose the appro-
priate enzyme and substrate concentrations as well as the enzyme
ratios. We therefore identified and determined the relevant kinetic
parameters for all the three enzymes. The kinetic constants were
determined by rearranging the non-linear rate responses of the
enzymes as a function of concentrations into a linear form using
double reciprocal (Lineweaver–Burk) and its secondary plot meth-
ods. Table 3 shows the kinetic properties and values of the kinetic
constants for RAMA, FDH and sn-G3PDH with respect to their native
substrates and products.
aldol cleavage of d-F16BP, as it is reduced in-situ to sn-G3P. RAMA
is non-competitively inhibited by d-GAP. The smaller competitive
inhibition constant (Kic,d-GAP) compared to the un-competitive inhi-
bition constant (Kiu,d-GAP) suggests that d-F16BP and d-GAP impede
each other in forming a complex with the active site of RAMA.
The kinetic models were validated by simulating the time course
of several batch conversions at different starting substrate and
enzyme concentrations using Matlab®. Evaluations of the exper-
imental data and the numerically simulated data matrices show
for the models of FDH and RAMA a 98.8% and 98.5% 2-D corre-
lation, respectively. Graphical correlations between experimental
and numerically simulated conversions versus reaction time for
both models are shown in Fig. 4.
ꢀ
ꢁ
NAD+
ꢂ
ꢃ
ꢀ
ꢁ
v1 = vmax
×
Km,NAD+ × 1 + NADH /K
+
NAD+
[
]
ic,NADH
ꢀ
ꢁ
HCOO−
ꢀ
ꢁ
×
(1)
Km,HCOO− + HCOO−
The overall model for the reaction kinetics can be developed by
fixing one of the reaction steps as rate limiting. The rate limiting
step can be adjusted by knowing the kinetics behavior of each of
the enzymes and choosing an appropriate enzyme ratio. In our case
the RAMA-catalyzed aldol cleavage of d-F16BP was set to be the
rate limiting step for the purpose of simplification and to avoid the
accumulation of d-GAP in the reaction solution. It is thus essential
to maintain the sn-G3PDH-catalyzed reduction of DHAP at a higher
rate than the RAMA-catalyzed aldol cleavage of d-F16BP in order
to suppress the reverse aldol condensation reaction and to pre-
The mathematical models shown in Eqs. (1) and (2) were devel-
oped for FDH and RAMA kinetics, respectively. The effect of DHAP
was not considered in the rate equation for the RAMA-catalyzed

80
G.S. Molla et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 77–82
Fig. 5. Comparison of stirred tank reactor (STR) and continuously operated stirred
reactor (CSTR) based on conversion as a function of reaction time for STR or residence
time for CSTR for the syntheses of d-GAP and sn-G3P: 5 mM d-F16BP, 0.5 mM NADH,
50 mM NaHCOO, 0.15 mg/ml RAMA, 1.43 mg/ml sn-G3PDH, 3.7 mg/ml FDH in 50 mM
TEA buffer pH 8 and 25 ◦C.
Fig. 6. Comparison of stirred tank reactor (STR) and continuously operated stirred
reactor (CSTR) based on selectivity as a function conversion for the syntheses of d-
GAP and sn-G3P: 5 mM d-F16BP, 0.5 mM NADH, 50 mM NaHCOO, 0.15 mg/ml RAMA,
1.43 mg/ml sn-G3PDH, 3.7 mg/ml FDH in 50 mM TEA buffer pH 8 and 25 ◦C.
Fig. 5 shows a comparison of the batch-wise operation in a
stirred tank reactor (STR) and the continuous operation in a contin-
uously operated stirred tank reactor (CSTR) based on conversion.
Conversion is defined as the number of d-F16BP molecules con-
verted per starting d-F16BP molecules. As can be seen in Fig. 5
higher conversion in a short time can be achieved via STR, e.g., in
vent the effect of DHAP on the activity of RAMA. Additionally, the
FDH-catalyzed NADH regeneration must be adjusted to a higher
rate than the sn-G3PDH-catalyzed reduction of DHAP so that sn-
G3PDH activity is not affected by NADH shortage. Therefore, the
overall reaction kinetics is simplified to a uni-uni irreversible enzy-
matic reaction and can be defined using the reaction kinetics model
developed for RAMA shown in the following Eq. (2).
D − F16BP
ic,D−GAP
[
]
ꢂ
ꢃ
ꢂ
ꢃ
v2 = vmax
×
(2)
Km,D−F16BP
×
1 + D − GAP /K
+ D − F16BP × 1 + D − GAP /K
[
]
[
]
[
]
iu,D−GAP
order to achieve a comparable conversion of 95% in the same reac-
tion time for STR and residence time for CSTR, the CSTR requires a
20-fold higher amount of enzyme than the STR.
2.3. Selection of an appropriate reactor type and separation
method development
Fig. 6 shows that the STR shows a better selectivity perfor-
mance than the CSTR. Selectivity is defined as the number of d-GAP
molecules synthesized per number of d-F16BP molecules con-
verted. For the synthesis of d-GAP, selectivity is a useful parameter
due to the exponential decay of d-GAP with time at pH 8. The selec-
tivity performance of the reactors was evaluated as a function of
conversion using the same amount of enzymes.
The performance of different reactor modes of operation was
evaluated by combining the developed reaction kinetics model,
mass balances of reactors and kinetics of the non-enzymatic
decomposition of d-GAP. The performance of the reactors was
evaluated using the parameters such as conversion, selectivity,
space-time yield (STY) and specific productivity. Eqs. (3) and (4)
show differential equations for the simulation of d-F16BP and d-
GAP concentrations for batch-wise mode of operation in a stirred
tank reactor (STR), respectively. Eqs. (5) and (6) show steady state
differential equations for the simulation of d-F16BP and d-GAP con-
centrations for a continuous mode of operation in a continuously
operated stirred tank reactor (CSTR), respectively. D − F16BP o
Inhibition of RAMA by d-GAP leads in the case of the CSTR
to a low steady-state reaction rate. Using conditions given in
Figs. 5 and 6 at the same conversion of 95% STR offers STY of
10.56 gL−1 day−1 while the STY of CSTR drops to 0.08 gL−1 day−1
due to their selectivity difference. Therefore, the most convenient
reactor type for the one-pot cascade enzymatic syntheses of d-GAP
and sn-G3P is the STR, due to the gradual increase of product with
reaction time. The separation of d-GAP and sn-G3P was achieved by
using TLC containing the strong basic anion exchanger polyethylen-
imine (PEI)-cellulose and by considering d-GAP stability in the
optimization of developing solvent and pH. A solution of 1 M KCl
dissolved in 100 mM HCl pH 2 was selected for high separation
ability and stability of d-GAP. An excellent separation of d-GAP (Rf
of 0.2) and sn-G3P (Rf of 0.9) has been achieved. Up-scaling the
TLC separation methodology to preparative column chromatog-
raphy scale is however hindered by a current commercial none
availability of the polyethylenimine (PEI)-cellulose material. Prepa-
ration of polyethylenimine (PEI)-cellulose material is therefore in
the interest of our research in order to scale-up the designed reac-
tion sequence to preparative scale.
[
]
and D − F16BP represent influx and efflux concentrations in CSTR,
[
]
respectively. D − GAP o and D − GAP represent influx into the
[
]
[
]
CSTR, which is negligible, and efflux, respectively. As can be seen,
Eqs. (4) and (6) include the kinetics of d-GAP decay at the reaction
conditions assuming the unit of reaction time and residence time
in hour.
∂ D − F16BP
[
]
= RAMA −v
(3)
(4)
(5)
[
] (
)
2
∂t
∂ D − GAP
[
]
= RAMA
v
2
× e−0.083t
[
] (
)
∂t
∂ D − F16BP
D − F16BP o − D − F16BP
])
[
]
([
]
[
=
− RAMA v
[
]
2
ꢀ
∂t
ꢄ
ꢅ
∂ D − GAP
D − GAP o − D − GAP
[
]
([
]
[
])
=
+ RAMA v
[
]
2
ꢀ
∂t
3. Conclusions
× e−0.083ꢀ
(6)
A one-pot enzymatic reaction sequence has been designed for
preparing d-GAP and sn-G3P. The reaction sequence shows a sig-

G.S. Molla et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 77–82
81
nificant improvement of the RAMA-catalyzed aldol cleavage of
d-F16BP yielding 100% conversion of d-F16BP. Detailed character-
izations of the enzymatic reaction sequence were performed. The
degradation rates of d-GAP in 50 mM TEA buffer at pH 8 and 25 ◦C
was measured. First order kinetic rate constant of 2.3 × 10−5 s−1 and
half-life of 8.35 h have been determined. d-GAP has been found to
show higher stability in Tris–HCl buffer.
a function of pH. The reactions were started by the addition of
required amount of sn-G3PDH followed by 0.004 mg/ml of RAMA
so that RAMA catalyzed aldol cleavage of d-F16BP is rate limiting. A
substrate solution of 0.5 mM DHAP and 0.5 mM NADH was prepared
in 50 mM TEA buffer and used to assay the activity of sn-G3PDH
as a function of pH. All reactions were started by the addition of
0.8 U/ml of sn-G3PDH. For the activity assay of FDH as a function of
pH, a substrate solution of 50 mM NaHCOO and 0.5 mM NAD+ was
prepared in 50 mM TEA buffer and all reactions were started by the
addition of 0.075 mg/ml of FDH. The long term operational stabil-
ities of RAMA, sn-G3PDH and FDH were examined by incubating
the enzymes in 50 mM TEA buffer at different pH from 5 to 9 and
25 ◦C. The remaining activities were routinely analyzed with the
assays described above for the activity measurements as a function
of pH. The stability of d-GAP was examined by incubating 20 mM
of d-GAP prepared in non-buffered medium, 100 mM TEA, 50 mM
TEA, 100 mM Tris–HCl and 100 mM PPB buffer pH 8 at 25 ◦C. The
degradation of d-GAP was analyzed via HPLC.
The capability of the reaction sequence to shift the equilibrium
was demonstrated by performing a batch reaction using a sub-
strate solution of 0.25 mM d-F16BP and 0.5 mM NADH prepared
in 50 mM TEA buffer pH 8. The reaction was started by the addi-
tion of 0.044 mg/ml of sn-G3PDH and 0046 mg/ml of RAMA with
respective order. Two batch reactions using 0.25 mM and 0.5 mM
d-GAP, both containing 0.5 mM NADH, for checking the selectiv-
ity of sn-G3PDH toward d-GAP and sn-G3P were carried out in
50 mM TEA pH 8 and 25 ◦C. Activity measurements as function of
co-substance concentrations were carried out in 50 mM TEA pH
8 and 25 ◦C. Linear representations of Michaelis–Menten enzyme
kinetics equation were applied in order to determine the values
of kinetics parameters. Separation of d-GAP and sn-G3P by using
polyethylenimine (PEI)-cellulose strong basic anion exchange TLC
plate was performed using 1 M KCl dissolved in 100 mM HCl pH
2 as a developing solvent. A staining solution containing 0.01 g/ml
of ammonium molybdate, 0.01 g/ml of ascorbic acid and 0.01 g/ml
of anthranilic acid was prepared in 65% nitric acid:methanol (1:9,
V/V) using a method as described elsewhere [45].
Each of the three enzymes involved in the reaction sequence
was investigated regarding kinetic behavior in order to choose
the appropriate enzyme and substrate concentrations and enzyme
ratios. RAMA is non-competitively inhibited by d-GAP and the
competitive and un-competitive inhibition constants suggest that
d-F16BP and d-GAP impede each other for complexation with the
active site of RAMA. The reaction kinetics models were validated
by simulating the time course of several batch reactions. The per-
formance of different reactor modes of operation for the enzymatic
reaction sequence was evaluated by combining the developed reac-
tion kinetics model, mass balances of reactors and kinetics of the
non-enzymatic decomposition of d-GAP. Batch-wise operation in
a STR is the most convenient process for the one-pot enzymatic
syntheses of d-GAP and sn-G3P. Alternative to STR, continuous
operation using packed bed reactor (PBR) can be applied due to the
gradual increase of the inhibitory product across the length of the
reactor. However, in this study, homogenous soluble enzymes were
used and a strategy to use three immobilized enzymes often shows
low efficiency due to mass transport limitations. The separation
of d-GAP and sn-G3P has been achieved using polyethylenimine
(PEI)-cellulose TLC. Preparation of polyethylenimine (PEI)-cellulose
material is in the interest of our research in order to scale-up the TLC
separation methodology to preparative column chromatography
scale.
4. Experimental
4.1. Materials
Ammonium molybdate, anthranilic acid, ascorbic acid,
d-fructose 1,6-bisphosphate (d-F16BP) d-glyceraldehyde 3-
phosphate, sn-glycerol 3-phosphate bis(cyclohexylammonium)
salt, reduced form of ␤-nicotinamide adenine dinucleotide dis-
odium salt, oxidized form of ␤-nicotinamide adenine dinucleotide
disodium salt, sodium formate, fructose-bisphosphate aldolase
from rabbit muscle (RAMA), sn-glycerol 3-phosphate dehydro-
genase (sn-G3PDH) from rabbit muscle, formate dehydrogenase
from C. boidinii (FDH), were purchased from Sigma–Aldrich
GmbH (Buchs, Switzerland). Nitric acid (65%), potassium chloride,
hydrochloric acid, potassium dihydrogen phosphate, methanol and
triethanolamine were purchased from Carl Roth GmbH (Karlsruhe,
Germany). All chemicals and solvents were used without further
purification. Polyethylenimine (PEI) cellulose strong basic anion
exchange TLC plate was purchased from Merck KGaA (Darmstadt,
Germany).
Acknowledgment
We would like to thank the German Federal Ministry of Educa-
tion and Research (BMBF) for financing the project P28 under the
cluster of Biocatalysis2021.
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.molcatb.2015.12.
004.
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