A Redox-Neutral, Two-Enzyme Cascade for the Production of Malate and Gluconate from Pyruvate and Glucose

Abstract

A triple mutant of NADP(H)-dependent malate dehydrogenase from thermotolerant Thermococcus kodakarensis has an altered cofactor preference for NAD+, as well as improved malate production compared to wildtype malate dehydrogenase. By combining mutant malate dehydrogenase with glucose dehydrogenase from Sulfolobus solfataricus and NAD+/NADH in a closed reaction environment, gluconate and malate could be produced from pyruvate and glucose. After 3 h, the yield of malate was 15.96 mM. These data demonstrate the feasibility of a closed system capable of cofactor regeneration in the production of platform chemicals.

Full text

applied
sciences
Article
A Redox-Neutral, Two-Enzyme Cascade for the Production of
Malate and Gluconate from Pyruvate and Glucose
Ravneet Mandair 1, Pinar Karagoz 1,2 and Roslyn M. Bill 1, *


Citation: Mandair, R.; Karagoz, P.;
Bill, R.M. A Redox-Neutral,
Two-Enzyme Cascade for the
Production of Malate and Gluconate
from Pyruvate and Glucose. Appl. Sci.
2021,11, 4877. https://doi.org/
10.3390/app11114877
Academic Editor: Francesca Scargiali
Received: 22 March 2021
Accepted: 24 May 2021
Published: 26 May 2021
Publisher’s Note: MDPI stays neutral
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1College of Health and Life Sciences, Aston University, Birmingham B4 7ET, UK;
ravmandair92@hotmail.com (R.M.); p.karagoz@ucl.ac.uk (P.K.)
2Department of Biochemical Engineering, University College London, Gower Street, London WC1E 6BT, UK
*Correspondence: r.m.bill@aston.ac.uk; Tel.: +44-12-1204-4274
Abstract:
A triple mutant of NADP(H)-dependent malate dehydrogenase from thermotolerant Ther-
mococcus kodakarensis has an altered cofactor preference for NAD
+
, as well as improved malate
production compared to wildtype malate dehydrogenase. By combining mutant malate dehydroge-
nase with glucose dehydrogenase from Sulfolobus solfataricus and NAD
+
/NADH in a closed reaction
environment, gluconate and malate could be produced from pyruvate and glucose. After 3 h, the
yield of malate was 15.96 mM. These data demonstrate the feasibility of a closed system capable of
cofactor regeneration in the production of platform chemicals.
Keywords:
enzymatic cascade reaction; malate production; malate dehydrogenase; glucose dehydrogenase
1. Introduction
Platform chemicals are molecules that can be used to make a variety of industrially
valuable products including solvents, polymers, pharmaceuticals and foods [
1
]. Most
commercial platform chemicals are mass-produced from petrochemical processes that use
fossil fuels as the starting raw materials [
2
], typically via low-cost, high-volume commodity
chemicals (methanol, ethylene, propene, butadiene, benzene, toluene and xylene) [
3
]. While
recent advances have enabled the use of renewable approaches on an industrial scale (such
as the production of polylactide and polyethylene from ethylene and biomass-derived lactic
acid [
4
]), the sustainable production of a wider range of platform chemicals is a priority
given global concerns about the environment, limited resources, and high oil prices [3,5].
One platform chemical in high global demand is malic acid. Malic acid, along with
other four-carbon diacids (such as succinic and fumaric acids), was reported as one of the
top value-added chemicals from biomass in a 2004 U.S. Department of Energy report [
6
].
Four-carbon diacids can be converted into 1,4-butanediol which can be further converted
into high-value products such as tetrahydrofuran, an important material for polymer
production. The current commercial production of malic acid is via the chemical hydration
of petrochemically derived maleic/fumaric acid [
7
], but advances in the field of metabolic
engineering have renewed interest in its bio-based production [
8
]. Natural high-capacity
malic acid producers such as Aspergillus flavus are not well-suited to industrial use due to
their physiological constraints [
3
] and the associated generation of aflatoxin (a carcinogen).
A particular concern is the need to control multiple metabolic pathways that lead to the
formation of unwanted intermediates [9], thereby reducing conversion efficiencies.
Cell-free metabolic engineering uses purified enzyme systems or crude cell lysates
in the synthesis of complex biomolecular commodities, thereby overcoming limitations
associated with using whole cells [
3
]. However, the high cost of processes involving
enzyme purification and the need to supplement reactions with the necessary cofactors
are current challenges. The nicotinamide nucleotides NAD
+
and NADP
+
are used by most
redox enzymes, and their balance is crucial in cell-free enzymatic pathways which lack
metabolic regulation from living cells [
3
]. Developing cascades consisting of multiple
Appl. Sci. 2021,11, 4877. https://doi.org/10.3390/app11114877 https://www.mdpi.com/journal/applsci

Appl. Sci. 2021,11, 4877 2 of 11
enzymes in cell-free pathways is a recent development [
10
]. This has led to developments
in the design of cell-free systems in which cofactors can be recycled on account of their
high costs [
11
]. Glucose dehydrogenase (GDH), along with formate dehydrogenase (FDH),
are the two most widely used enzymes for cofactor recycling in industrial/commercial
processes, with GDH from the Bacillus species showing the highest activity and stability,
leading to its wide applications in industry [12].
NADP(H)-dependent malate dehydrogenase from the thermophilic archaeon Thermo-
coccus kodakarensis, catalyzes the HCO
3−
independent reductive carboxylation of pyruvate
to malate and produces lactate as a by-product. Its application in artificial enzymatic
cascades that use NADH as a cofactor is now possible following directed evolution to alter
its cofactor preference from NADPH to NADH [
13
]. A triple mutant was identified that
exhibited a sixfold higher k
cat
/K
M
for NAD
+
than the wildtype enzyme together with
increased malate (and lower lactate) yields. The triple mutant enzyme (hereafter referred
to as MDH*) catalyzed pyruvate carboxylation with NADH to give a 1.2 times higher yield
of malate than the wildtype enzyme with NADPH [13].
We previously immobilized purified recombinant GDH from Sulfolobus solfataricus
onto novel, hierarchically structured macroporous–mesoporous silica supports and pro-
duced gluconic acid from both glucose and bread waste hydrolysate using NAD
+
as a
cofactor. The aim of the present study was to use GDH and MDH* in a closed system
capable of cofactor regeneration in the production of malate from pyruvate (Supplemen-
tary Materials, Figure S1). We hypothesized that MDH* would use the cofactor byproduct
of GDH-facilitated gluconic acid production from glucose to catalyze the conversion of
pyruvate to malate via NADH, leading to its regeneration in the process.
2. Materials and Methods
2.1. Microorganisms and Enzyme Production
Expression—the gene encoding MDH* from Thermococcus kodakarensis (GenBank ac-
cession number BAE47514.1) was synthesized and cloned into pET-30a via NdeI and BamHI
at the 5
0
and 3
0
restriction sites, respectively. The full-length gene encoding 3 mutations
(R221G, K228R and I310V) was synthesized by GenScript with the sequence optimized
for expression in E. coli and the vector was transformed into BL21-DE3 competent cells.
Cells were grown in LB media supplemented with 50
µ
g/mL kanamycin and grown
at 37
◦
C and 220 rpm to A
600
= 0.6. 1 mM (final concentration) IPTG (isopropyl ß-D-1-
thiogalactopyranoside) was used for induction and the cells were grown at room tempera-
ture for 18 h at 220 rpm.
Purification—cell pellets were obtained by centrifugation and re-suspended in 100 mM
HEPES pH 7.5, 200 mM MgCl
2
, 10% glycerol. Cell lysis was carried out using a cell disrup-
tor system (Bandelin, SONOPULS, Berlin, Germany), followed by heat treatment at 70
◦
C
for 30 min. Cellular debris was removed by centrifugation, and the crude lysate was bound
overnight to Ni–NTA resin (Qiagen, Hilden, Germany) and eluted with buffer containing
500 mM imidazole, 100 mM HEPES pH 7.5, 200 mM MgCl
2
, 10% glycerol. The protein
was then dialyzed into 100 mM HEPES pH 7.5, 200 mM MgCl
2
, 10% glycerol. Fractions
containing MDH* were pooled and their protein concentration was determined using
the bicinchoninic acid assay (BCA; Reagent Compatible BCA Assay Kit, ThermoFisher
Scientific, Waltham, MA, USA) and a Nanodrop device (ThermoFisher Scientific, Waltham,
MA, USA). Samples were snap-frozen in liquid nitrogen and stored at
−
80
◦
C. GDH used
in this study was expressed, purified, and characterized as previously described [14].
2.2. Enzyme Activity Assays
MDH* activity was assayed in 500 mM HEPES pH 7.5, 5 mM NH
4
Cl, 0.5 mM MnCl
2
,
30 mM pyruvate, 85 mM NaHCO
3
and 1 mM NADH. Unless otherwise stated, the activity
assay for MDH* was conducted in 96-well plates in a FLUOstar Omega Microplate Reader
fitted with an atmospheric control unit (allowing full control and regulation of oxygen and
carbon dioxide within the microplate reader chamber) at 55
◦
C for 2 h at 15% CO
2
and

Appl. Sci. 2021,11, 4877 3 of 11
5% O
2
. The activity of both MDH* and GDH (produced as described previously [
14
]) was
detected by monitoring the NAD
+
concentrations of the supernatants with a plate reader
at 340 nm. Optimization studies were performed using MATLAB 2019a.
2.3. Determination of Kinetic Constants
The kinetic constants for MDH* were determined by measuring the reaction rates at
regular time intervals and different substrate levels (0–75 mM). K
M
and V
max
values were
calculated from Lineweaver–Burk plots.
2.4. HPIC Analysis
Known standard concentrations of substrates/products were analyzed via HPIC to
produce standard curves. These standard curves were then used to calculate the unknown
concentrations of chemicals of interest. HPIC was performed using a Dionex
™
Integrion
™
HPIC
™
System (Thermo Fisher Scientific, Waltham, MA, USA). Unless otherwise stated,
all samples were diluted ten times and detected under reaction conditions of 5 mM eluent
(NaOH) at 30
◦
C at a 0.38 mL/min flow rate. It was not possible to analyze glucose,
gluconate or lactate with HPIC, and alternative chemical and enzymatic approaches were
insufficiently sensitive. Pyruvate and malate were detectable in 2 mL reactions.
3. Results
3.1. Purity and Activity of Recombinant MDH*
In building an enzyme cascade, the use of thermophilic enzymes has the potential
to improve enzyme stability and therefore reduce costly enzyme purification. As we
previously demonstrated, recombinant thermophilic GDH can be purified in a single
step after initial heat treatment at 70
◦
C for 30 min [
14
]. In this study, we also produced
recombinant thermophilic MDH* in order to couple its activity with that of GDH. Following
heat treatment and affinity purification, MDH*-containing fractions were analyzed by SDS-
PAGE to determine protein purity. Figure 1shows that purified recombinant MDH* (which
contained a decahistidine tag and linker with a combined molecular weight of 50 kDa)
was >95% pure. Protein-containing fractions were analyzed by BCA, pooled, and used
immediately in experiments or snap-frozen prior to storage at −80 ◦C.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 4 of 12
Figure 1. SDS-PAGE analysis of purified MDH*. Lane 1 shows molecular markers, and lane 2 is
MDH* purified by Ni–NTA affinity chromatography.
3.2. Effect of GDH Buffer Components on the Activity of MDH*
In order to identify reaction conditions that would support both enzymes in a one-
pot cascade reaction, it was necessary to analyze the effect on MDH* activity of buffer
components required for optimum GDH activity. The assay reagents needed for the two
different enzymes varied, with some being used at different concentrations whilst others
are only needed for one enzyme and not the other (Table 1). In particular, MDH* activity
assays require additional assay reagents in comparison to GDH, as well as atmospheric
control (Table 1).
Table 1. Standard assay conditions for GDH and MDH*.
GDH Standard Assay Conditions at 55 °C MDH* Standard Assay Conditions at 55 °C
No atmospheric control required
100 mM HEPES pH 7.5
2.5% glycerol
30 mM MgCl2
100 mM glucose
5 mM NAD+
15% CO2 and 5% O2
500 mM HEPES pH 7.5
5 mM NH4Cl
0.5 mM MnCl2
30 mM pyruvate
85 mM NaHCO3
1 mM NADH
The most significant effect was seen when the HEPES concentration was reduced
from 500 mM to 100 mM (Figure 2), which is the concentration used in the GDH buffer
(Table 1). As a result, the activity of MDH* reduced by 20% (Figure 2). In contrast, the
addition of glycerol, glucose, and MgCl2 into the MDH* reaction mixture had no
significant effect on MDH* enzymatic activity.
Figure 1.
SDS-PAGE analysis of purified MDH*. Lane 1 shows molecular markers, and lane 2 is
MDH* purified by Ni–NTA affinity chromatography.

Appl. Sci. 2021,11, 4877 4 of 11
3.2. Effect of GDH Buffer Components on the Activity of MDH*
In order to identify reaction conditions that would support both enzymes in a one-
pot cascade reaction, it was necessary to analyze the effect on MDH* activity of buffer
components required for optimum GDH activity. The assay reagents needed for the two
different enzymes varied, with some being used at different concentrations whilst others
are only needed for one enzyme and not the other (Table 1). In particular, MDH* activity
assays require additional assay reagents in comparison to GDH, as well as atmospheric
control (Table 1).
Table 1. Standard assay conditions for GDH and MDH*.
GDH Standard Assay Conditions at 55 ◦C MDH* Standard Assay Conditions at 55 ◦C
No atmospheric control required
100 mM HEPES pH 7.5
2.5% glycerol
30 mM MgCl2
100 mM glucose
5 mM NAD+
15% CO2and 5% O2
500 mM HEPES pH 7.5
5 mM NH4Cl
0.5 mM MnCl2
30 mM pyruvate
85 mM NaHCO3
1 mM NADH
The most significant effect was seen when the HEPES concentration was reduced from
500 mM to 100 mM (Figure 2), which is the concentration used in the GDH buffer (Table 1).
As a result, the activity of MDH* reduced by 20% (Figure 2). In contrast, the addition of
glycerol, glucose, and MgCl
2
into the MDH* reaction mixture had no significant effect on
MDH* enzymatic activity.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 12
Figure 2. Effect of GDH assay buffer components on the activity of MDH*. Control is the standard
MDH* assay buffer (Table 1); the second bar represents the Control with a decreased HEPES
buffer concentration (100 mM rather than 500 mM HEPES); the third bar represents the Control
with the addition of 30 mM MgCl2; the fourth bar represents the Control with the addition of 2.5%
glycerol; the fifth bar represents the Control with the addition of 100 mM glucose.
3.3. Effect of Atmospheric Conditions Required by MDH* on the Activity of GDH
The activity of GDH was tested at 55 °C, 15% CO2, and 5% O2 atmospheric conditions
in a standard GDH reaction mix. The activity of the GDH under these conditions was
compared with the activity of GDH under its standard aerobic (without atmospheric
control) assay conditions. Activity of GDH was determined not to be affected by the
atmospheric conditions required by MDH* (Figure 3).
Figure 3. Activity of GDH under aerobic and 15% CO2 and 5% O2 atmospheric condition (results
are the averages of 3 different readings, standard deviation for all conditions ≤0.07).
Figure 2.
Effect of GDH assay buffer components on the activity of MDH*. Control is the standard
MDH* assay buffer (Table 1); the second bar represents the Control with a decreased HEPES buffer
concentration (100 mM rather than 500 mM HEPES); the third bar represents the Control with the
addition of 30 mM MgCl
2
; the fourth bar represents the Control with the addition of 2.5% glycerol;
the fifth bar represents the Control with the addition of 100 mM glucose.
3.3. Effect of Atmospheric Conditions Required by MDH* on the Activity of GDH
The activity of GDH was tested at 55
◦
C, 15% CO
2
, and 5% O
2
atmospheric conditions
in a standard GDH reaction mix. The activity of the GDH under these conditions was
compared with the activity of GDH under its standard aerobic (without atmospheric

Appl. Sci. 2021,11, 4877 5 of 11
control) assay conditions. Activity of GDH was determined not to be affected by the
atmospheric conditions required by MDH* (Figure 3).
Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 12
Figure 2. Effect of GDH assay buffer components on the activity of MDH*. Control is the standard
MDH* assay buffer (Table 1); the second bar represents the Control with a decreased HEPES
buffer concentration (100 mM rather than 500 mM HEPES); the third bar represents the Control
with the addition of 30 mM MgCl2; the fourth bar represents the Control with the addition of 2.5%
glycerol; the fifth bar represents the Control with the addition of 100 mM glucose.
3.3. Effect of Atmospheric Conditions Required by MDH* on the Activity of GDH
The activity of GDH was tested at 55 °C, 15% CO2, and 5% O2 atmospheric conditions
in a standard GDH reaction mix. The activity of the GDH under these conditions was
compared with the activity of GDH under its standard aerobic (without atmospheric
control) assay conditions. Activity of GDH was determined not to be affected by the
atmospheric conditions required by MDH* (Figure 3).
Figure 3. Activity of GDH under aerobic and 15% CO2 and 5% O2 atmospheric condition (results
are the averages of 3 different readings, standard deviation for all conditions ≤0.07).
Figure 3.
Activity of GDH under aerobic and 15% CO
2
and 5% O
2
atmospheric condition (results are
the averages of 3 different readings, standard deviation for all conditions ≤0.07).
3.4. Effect of MDH* Assay Buffer Components on the Activity of GDH
Having examined the effect of GDH buffer components on MDH* activity, the effects
of MDH* buffer components were examined on the activity of GDH under 15% CO
2
and
5% O
2
. The initial glucose concentration was 100 mM. Figure 4shows that the presence of
pyruvate has a significant effect on the activity of GDH. When the pyruvate concentration
was 30 mM, GDH activity decreased by 96.7%. Increasing the final concentration of HEPES
from 100 mM to 500 mM decreased the activity of GDH by 15.5%. When MnCl
2
and
NaHCO
3
were added to the buffer, the activity of GDH showed an increase of 6.9% and
8.2%, respectively.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 12
3.4. Effect of MDH* Assay Buffer Components on the Activity of GDH
Having examined the effect of GDH buffer components on MDH* activity, the effects
of MDH* buffer components were examined on the activity of GDH under 15% CO2 and
5% O2. The initial glucose concentration was 100 mM. Figure 4 shows that the presence of
pyruvate has a significant effect on the activity of GDH. When the pyruvate concentration
was 30 mM, GDH activity decreased by 96.7%. Increasing the final concentration of
HEPES from 100 mM to 500 mM decreased the activity of GDH by 15.5%. When MnCl2
and NaHCO3 were added to the buffer, the activity of GDH showed an increase of 6.9%
and 8.2%, respectively.
Figure 4. Effect of MDH* assay buffer components on the activity of GDH. Control is the standard
GDH assay buffer (Table 1); the second bar represents the Control with 500 mM instead of 100 mM
HEPES; the third bar represents the Control with the addition of 5 mM NH4Cl; the fourth bar
represents the Control with the addition of 0.5 mM MnCl2; the fifth bar represents the Control
with the addition of 85 mM NaHCO3; the sixth bar represents the Control with the addition of 30
mM pyruvate.
3.5. Effect of Pyruvate Concentration on the Activity of GDH
The presence of pyruvate had a substantial negative effect on the activity of GDH
(Figure 4). The effect of adding pyruvate at concentrations of 0–60 mM to the standard
GDH assay buffer was therefore examined. NADH formation was recorded over 2.5 h and
the relative activity of GDH was calculated (Figure 5). As shown in Figure 5, pyruvate
concentrations above 10 mM had a significant effect on the activity of GDH and reduced
it by over 50%. This was further explored by following NADH production in the first hour
of the experiment with pyruvate concentrations ranging from 0–30 mM. Figure 6 shows
the effect of different pyruvate concentrations on the activity of the GDH during the first
hour of the experiment, whereby concentrations of <1 0 mM of pyruvate had no significant
effect on the reaction
Figure 4.
Effect of MDH* assay buffer components on the activity of GDH. Control is the standard
GDH assay buffer (Table 1); the second bar represents the Control with 500 mM instead of 100 mM
HEPES; the third bar represents the Control with the addition of 5 mM NH
4
Cl; the fourth bar
represents the Control with the addition of 0.5 mM MnCl
2
; the fifth bar represents the Control
with the addition of 85 mM NaHCO
3
; the sixth bar represents the Control with the addition of
30 mM pyruvate.

Appl. Sci. 2021,11, 4877 6 of 11
3.5. Effect of Pyruvate Concentration on the Activity of GDH
The presence of pyruvate had a substantial negative effect on the activity of GDH
(Figure 4). The effect of adding pyruvate at concentrations of 0–60 mM to the standard
GDH assay buffer was therefore examined. NADH formation was recorded over 2.5 h and
the relative activity of GDH was calculated (Figure 5). As shown in Figure 5, pyruvate
concentrations above 10 mM had a significant effect on the activity of GDH and reduced it
by over 50%. This was further explored by following NADH production in the first hour of
the experiment with pyruvate concentrations ranging from 0–30 mM. Figure 6shows the
effect of different pyruvate concentrations on the activity of the GDH during the first hour
of the experiment, whereby concentrations of <10 mM of pyruvate had no significant effect
on the reaction.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 12
Figure 5. Effect of pyruvate concentration of the activity of GDH.
Figure 6. Effect of pyruvate concentration on NADH production by GDH using 100 mM glucose
as a substrate (results are the averages of 3 different readings, standard deviation for all conditions
≤0.03).
3.6. Optimization of Atmospheric and Temperature Conditions for the Activity of Purified
MDH*
The reaction conditions for the efficient conversion of pyruvate to malate by MDH*
require high CO2 and low O2 levels. All experiments undertaken were therefore
performed under standard MDH* reaction conditions (55 °C, 5% O2 and 15% CO2). This
was further explored and optimized by testing the effects of different O2 and CO2 ratios at
different temperatures on the activity of MDH* to obtain optimal reaction conditions. A
Box–Behnken design and response surface methodology were used with MATLAB 2019a,
to develop a model that identified the conditions leading to maximum MDH* activity.
The values of the three chosen input variables are shown in Supplementary Table S1. The
chosen temperature range of 45–65 °C was selected on account of instrumental technical
Figure 5. Effect of pyruvate concentration of the activity of GDH.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 12
Figure 5. Effect of pyruvate concentration of the activity of GDH.
Figure 6. Effect of pyruvate concentration on NADH production by GDH using 100 mM glucose
as a substrate (results are the averages of 3 different readings, standard deviation for all conditions
≤0.03).
3.6. Optimization of Atmospheric and Temperature Conditions for the Activity of Purified
MDH*
The reaction conditions for the efficient conversion of pyruvate to malate by MDH*
require high CO2 and low O2 levels. All experiments undertaken were therefore
performed under standard MDH* reaction conditions (55 °C, 5% O2 and 15% CO2). This
was further explored and optimized by testing the effects of different O2 and CO2 ratios at
different temperatures on the activity of MDH* to obtain optimal reaction conditions. A
Box–Behnken design and response surface methodology were used with MATLAB 2019a,
to develop a model that identified the conditions leading to maximum MDH* activity.
The values of the three chosen input variables are shown in Supplementary Table S1. The
chosen temperature range of 45–65 °C was selected on account of instrumental technical
Figure 6.
Effect of pyruvate concentration on NADH production by GDH using 100 mM glucose as a
substrate (results are the averages of 3 different readings, standard deviation for all
conditions ≤0.03
).

Appl. Sci. 2021,11, 4877 7 of 11
3.6. Optimization of Atmospheric and Temperature Conditions for the Activity of Purified MDH*
The reaction conditions for the efficient conversion of pyruvate to malate by MDH*
require high CO
2
and low O
2
levels. All experiments undertaken were therefore performed
under standard MDH* reaction conditions (55
◦
C, 5% O
2
and 15% CO
2
). This was further
explored and optimized by testing the effects of different O
2
and CO
2
ratios at different
temperatures on the activity of MDH* to obtain optimal reaction conditions. A Box–
Behnken design and response surface methodology were used with MATLAB 2019a, to
develop a model that identified the conditions leading to maximum MDH* activity. The
values of the three chosen input variables are shown in Supplementary Table S1. The
chosen temperature range of 45–65
◦
C was selected on account of instrumental technical
specifications and the use of a thermophilic enzyme. Fifteen independent experiments
were performed to examine the effect of the experimental variables on the initial reaction
rate of MDH* activity. The initial reaction rate was calculated via the decrease in NADH
concentration at the beginning of the reaction. The full data set for all 15 conditions is shown
in Table 2(and Supplementary Table S2). The highest initial reaction rate, 0.103 mM/min,
was detected at 65
◦
C, 7.5% O
2
, and 10% CO
2
for experiment number eight (Table 2). The
maximum initial reaction rate value was calculated by the model to be 0.102 mM/min,
with temperature, O
2
and CO
2
values of 65
◦
C, 5.625% and 12.5%, respectively. The
experimentally obtained value and the value as calculated by the model were in close
agreement. These conditions were then used for all further activity assays (65
◦
C, 5.6% O
2
and 12.5% CO2).
Table 2.
Experimental conditions and initial MDH* activity rates obtained with the input conditions
outlined in Supplementary Table S2 and as calculated using the model *.
Experiment
Number O2(%) CO2(%) Temperature
(◦C)
Initial Rate
(mM/min)
Initial Rate *
(mM/min)
1 2.5 5 55 0.068 0.066
2 7.5 5 55 0.061 0.064
3 2.5 15 55 0.067 0.065
4 7.5 15 55 0.086 0.088
5 2.5 10 45 0.030 0.039
6 7.5 10 45 0.059 0.063
7 2.5 10 65 0.098 0.095
8 7.5 10 65 0.103 0.094
9 5 5 45 0.052 0.045
10 5 15 45 0.054 0.082
11 5 5 65 0.076 0.049
12 5 15 65 0.092 0.099
13 5 10 55 0.078 0.086
14 5 10 55 0.092 0.086
15 5 10 55 0.088 0.086
3.7. Coupling MDH* and GDH for Cofactor Recycling
Having identified buffer conditions that could support the activity of both enzymes,
the activities and cofactor recycling capabilities of purified MDH* and GDH were assayed
within the same experiment at the optimal conditions of 65
◦
C, 5.6% O
2
and 12.5% CO
2
.
Pyruvate and glucose concentrations were each adjusted to 30 mM, the concentration of
HEPES was kept at 500 mM, and all other components were added to the reaction as
shown in Table 1. Two different experimental setups were used to determine the success of
coupling the two enzymes.

Appl. Sci. 2021,11, 4877 8 of 11
In the first setup, changes in pyruvate concentration were examined, which would
indicate the conversion of NADH to NAD
+
and pyruvate to malate by MDH*. The reaction
was tested under optimal conditions (65
◦
C, 5.6% O
2
and 12.5% CO
2
), by the addition of
NADH. In the second setup, the same reaction mixture was used but the reaction was
initiated by the addition of NAD
+
instead of NADH. Changes in the NADH concentration
were monitored for 2 h with a plate reader. The final NADH concentrations were compared
with an enzyme-free reaction control containing the same initial concentration of NADH as
the first experimental setup (Table 3). Despite the presence of 30 mM pyruvate, GDH still
displayed activity. The NADH consumption in the first setup demonstrated the activity of
MDH*. The concentrations of malate and gluconate produced by the cascade were also
established by deproteinizing samples of the reaction mixture, diluting them 10-fold, and
analyzing them via HPIC. However, due to the high dilution factor, malate peaks were
below the detection limit and pyruvate as the substrate was in excess; hence, small changes
in concentrations were indistinguishable. However, gluconic acid production was detected
in both cases, demonstrating that cofactor recycling had been achieved.
Table 3. Coupling GDH and MDH* for cofactor recycling capabilities.
Scenario 1: Presence
of Both Enzymes
(GDH and MDH*),
NADH as Cofactor
Scenario 2: Presence
of Both Enzymes
(GDH and MDH*),
NAD+as Cofactor
Control: No Enzyme,
Contains Same
Amount of NADH
as Scenario 1
Final NADH
concentration, mM 1.21 ±0.06 1.31 ±0.01 1.74 ±0.04
Gluconic acid
concentration, mM 8.81 10.45 0
To detect malate by HPIC, 2 mL cascade reactions were set up and monitored via
plate reader, comprising 13.2U GDH and 3.3U MDH* under optimal reaction conditions
(65
◦
C, 5.6% O
2
and 12.5% CO
2
). The reactions were initiated by the addition of NADH. All
experiments were carried out in triplicate under the same conditions for different periods
of time. An MDH*-only reaction was run as a control (Figure 7B). The gluconic and malic
acid yields from the cascade reaction were significantly higher than when MDH* was
used alone (Figure 7A). As a result, 15.96 mM malic acid and 9.94 mM gluconic acid were
produced by the cascade reaction after 3 h compared to 0.309 mM malic acid from the
MDH*-only reaction. Notably, 2 mM malic acid was produced by the dual enzyme system
after 15 min (which is higher than the malic acid yield by MDH* alone after a reaction time
of 3 h) and another high-value chemical, gluconic acid, was additionally produced by the
cascade system from glucose and NAD+.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 9 of 12
acid production was detected in both cases, demonstrating that cofactor recycling had
been achieved.
Table 3. Coupling GDH and MDH* for cofactor recycling capabilities.
Scenario 1: Presence of
Both Enzymes (GDH
and MDH*), NADH as
Cofactor
Scenario 2: Presence of
Both Enzymes (GDH
and MDH*), NAD+ as
Cofactor
Control: No Enzyme,
Contains Same Amount
of NADH as Scenario 1
Final NADH
concentration, mM 1.21 ± 0.06 1.31 ± 0.01 1.74 ± 0.04
Gluconic acid
concentration, mM 8.81 10.45 0
To detect malate by HPIC, 2 mL cascade reactions were set up and monitored via
plate reader, comprising 13.2U GDH and 3.3U MDH* under optimal reaction conditions
(65 °C, 5.6% O2 and 12.5% CO2). The reactions were initiated by the addition of NADH.
All experiments were carried out in triplicate under the same conditions for different
periods of time. An MDH*-only reaction was run as a control (Figure 7B). The gluconic
and malic acid yields from the cascade reaction were significantly higher than when
MDH* was used alone (Figure 7A). As a result, 15.96 mM malic acid and 9.94 mM gluconic
acid were produced by the cascade reaction after 3 h compared to 0.309 mM malic acid
from the MDH*-only reaction. Notably, 2 mM malic acid was produced by the dual
enzyme system after 15 min (which is higher than the malic acid yield by MDH* alone
after a reaction time of 3 h) and another high-value chemical, gluconic acid, was
additionally produced by the cascade system from glucose and NAD+.
(A)
Figure 7. Cont.

Appl. Sci. 2021,11, 4877 9 of 11
Appl. Sci. 2021, 11, x FOR PEER REVIEW 10 of 12
(B)
Figure 7. Substrate consumption and product formation by MDH* and GDH (A) and MDH* alone
(B).
4. Discussion
To ensure a successful coupled reaction, the effects of GDH (the regeneration
enzyme) reaction conditions on the activity of MDH* (the production enzyme) were
examined. The highest reduction in MDH* enzymatic activity was caused by a decrease
in HEPES buffer concentration from the standard 500 Mm HEPES in MDH* assays to 100
Mm HEPES in GDH assays; the activity of MDH* was reduced by 20% (Figure 2). The
majority of enzymes require only moderate ionic strength, typically ranging between 50
and 200 Mm, but this is not the case for thermophilic enzymes which require buffer
concentrations up to 1 M [15]. The observed decrease in activity is therefore likely due to
the reduction in buffer capacity which, along with any added components, can reduce the
obligatory enzyme activity [16]. Aside from HEPES, the addition of other GDH assay
components such as MgCl2, glycerol, and the GDH substrate, glucose, did not reduce
MDH* activity by more than 10% (Figure 2).
MDH* activity assays were carried out under 15% CO2 and 5% O2 atmospheric
conditions; therefore, GDH activity was assayed under atmospheric conditions required
by MDH* and activity levels of GDH were measured via the concomitant production of
NADH from the oxidation of glucose to gluconate. After 60 min, there was no significant
difference between the amount of NADH produced by GDH assayed under aerobic
conditions and GDH assayed under atmospheric control (Figure 3). From these findings,
it was concluded that GDH is a suitable cofactor recycling partner and its reaction
components do not drastically inhibit the activity of MDH*. It was concluded that GDH
can be utilized effectively under altered atmospheric conditions as per the requirements
of MDH*.
For a fully feasible cofactor recycling coupling, the assay components of MDH* were
also tested on the activity of GDH under atmospheric conditions. This experiment
demonstrated the drastic effect of pyruvate on reducing the activity of GDH (Figure 4).
This loss of activity was determined to be due to the competitive inhibition of GDH by
pyruvate, because concentrations of 10 mM pyruva te reduced GDH activity by ~60%, and
a twofold increase in pyruvate concentration increased the KM value sixfold
(Supplementary Table S3). Concentrations of 30 mM pyruvate and 30 mM glucose were
used to mitigate the issue of GDH inhibition by pyruvate. Cofactor recycling was
demonstrated when using NAD+ or NADH as cofactors in the presence of both GDH and
MDH* (Table 3).
Figure 7.
Substrate consumption and product formation by MDH* and GDH (
A
) and MDH* alone (
B
).
4. Discussion
To ensure a successful coupled reaction, the effects of GDH (the regeneration enzyme)
reaction conditions on the activity of MDH* (the production enzyme) were examined.
The highest reduction in MDH* enzymatic activity was caused by a decrease in HEPES
buffer concentration from the standard 500 Mm HEPES in MDH* assays to 100 Mm HEPES
in GDH assays; the activity of MDH* was reduced by 20% (Figure 2). The majority of
enzymes require only moderate ionic strength, typically ranging between 50 and 200 Mm,
but this is not the case for thermophilic enzymes which require buffer concentrations up to
1 M [
15
]. The observed decrease in activity is therefore likely due to the reduction in buffer
capacity which, along with any added components, can reduce the obligatory enzyme
activity [
16
]. Aside from HEPES, the addition of other GDH assay components such as
MgCl
2
, glycerol, and the GDH substrate, glucose, did not reduce MDH* activity by more
than 10% (Figure 2).
MDH* activity assays were carried out under 15% CO
2
and 5% O
2
atmospheric
conditions; therefore, GDH activity was assayed under atmospheric conditions required by
MDH* and activity levels of GDH were measured via the concomitant production of NADH
from the oxidation of glucose to gluconate. After 60 min, there was no significant difference
between the amount of NADH produced by GDH assayed under aerobic conditions and
GDH assayed under atmospheric control (Figure 3). From these findings, it was concluded
that GDH is a suitable cofactor recycling partner and its reaction components do not
drastically inhibit the activity of MDH*. It was concluded that GDH can be utilized
effectively under altered atmospheric conditions as per the requirements of MDH*.
For a fully feasible cofactor recycling coupling, the assay components of MDH* were
also tested on the activity of GDH under atmospheric conditions. This experiment demon-
strated the drastic effect of pyruvate on reducing the activity of GDH (Figure 4). This loss
of activity was determined to be due to the competitive inhibition of GDH by pyruvate, be-
cause concentrations of 10 mM pyruvate reduced GDH activity by ~60%, and a twofold in-
crease in pyruvate concentration increased the K
M
value sixfold (
Supplementary Table S3
).
Concentrations of 30 mM pyruvate and 30 mM glucose were used to mitigate the issue of
GDH inhibition by pyruvate. Cofactor recycling was demonstrated when using NAD
+
or
NADH as cofactors in the presence of both GDH and MDH* (Table 3).
In conclusion, we have demonstrated cofactor regeneration within a closed system of
two thermophilic enzymes. The work carried out here sets the foundation for this concept
to be explored further with longer reaction times and further optimization of reaction
conditions. The redox neutrality of the two enzymes may now be exploited further by
expanding the number of enzymes in the cascade system. Further optimization regarding
the separation of the products can also be explored. Immobilization of the coupled enzymes
will provide potential solutions to issues linked with enzyme cost and stability [
17
,
18
]

Appl. Sci. 2021,11, 4877 10 of 11
and for catalyst recycling on an industrial scale [
19
], offering advantages of both economy
and sustainability.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10
.3390/app11114877/s1, Figure S1: Schematic representation of redox balanced cascade with cofactor
recycling, Figure S2: Effect of atmospheric conditions and the reaction temperature on the initial
rate of MDH*, Table S1: Atmospheric condition independent variables and their values, Table S2:
Box-Behnken design for 3 factors with 3 center points, Table S3: Change in kinetic parameters of
GDH in the presence of different pyruvate concentrations.
Author Contributions:
Conceptualization, R.M., P.K. and R.M.B.; methodology, R.M. and P.K.;
formal analysis, R.M., P.K. and R.M.B.; resources, R.M.B.; writing—original draft preparation, R.M.,
P.K. and R.M.B.; writing—review and editing, R.M., P.K. and R.M.B.; funding acquisition, R.M.B. All
authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Biotechnology and Biological Sciences Research Council
(BBSRC) through the Global Challenges Research Fund Project, CAPRI-BIO (BB/P022685/1). R.M.
was supported by a BBSRC training grant with Chemoxy Ltd. (BB/M016668/1). The APC was
funded by BBSRC.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
NADP
+
—nicotinamide adenine dinucleotide phosphate; NAD
+
—nicotinamide adenine dinucleotide;
MDH*—triple mutant malate dehydrogenase; GDH—glucose dehydrogenase; FDH—formate de-
hydrogenase; IPTG—isopropyl ß-D-1-thiogalactopyranoside; MgCl
2
—magnesium chloride; Ni–
NTA—nickel nitrilotriacetic acid; BCA—bicinchoninic acid assay; NH
4
Cl—ammonium chloride;
MnCl
2
—manganese chloride; NaHCO
3
—sodium bicarbonate; HPIC—high-pressure ion chromatog-
raphy; SDS-PAGE—sodium dodecyl sulphate–polyacrylamide gel electrophoresis.
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