583
Regeneration of cofactors for use in biocatalysis
Huimin Zhao and Wilfred A van der Donky
Cofactor-dependent enzymes catalyze many synthetically useful
reactions. The high cost of cofactors, however, necessitates
in situ cofactor regeneration for preparative applications. After
two decades of research, several cofactors can now be
effectively regenerated using enzyme or whole-cell based
methods. Significant advances have been made in this area in
the past three years and include the development of novel or
improved methods for regenerating ATP, sugar nucleotides and
3-phosphoadenosine-50 -phosphosulphate. These approaches
have found novel applications in biocatalysis.
Addresses
Department of Chemical and Biomolecular Engineering, University
of Illinois at Urbana-Champaign, 600 S. Mathews Avenue,
Urbana, IL 61801, USA
e-mail: [email protected]
y
Department of Chemistry, University of Illinois at Urbana-Champaign,
600 S. Mathews Avenue, Urbana, IL 61801, USA
e-mail: [email protected]
Current Opinion in Biotechnology 2003, 14:583–589
This review comes from a themed issue on
Chemical biotechnology
Edited by Frances H Arnold and Anton Glieder
0958-1669/$ – see front matter
ß 2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2003.09.007
Abbreviations
AdK
ADP
AMP
ATP
CMP-NeuAc
CTP
GDP-Fuc
NTP
PAP
PAPS
PEP
PPK
TTN
UDP-Gal
UDP-GlcNAc
UTP
adenylate kinase
adenosine 50 -diphosphate
adenosine 50 -monophosphate
adenosine 50 -triphosphate
cytidine 50 -monophosphate N-acetylneuraminic acid
cytidine 50 -triphosphate
GDP-fucose
nucleoside triphosphate
polyphosphate:AMP phosphotransferase
3-phosphoadenosine-50 -phosphosulfate
phosphoenolpyruvate
polyphosphate kinase
total turnover number
UDP-galactose
UDP-N-acetylglucosamine
uridine 50 -triphosphate
Introduction
With recent advances in genomics and genetic engineering, the use of biocatalysis for industrial synthetic chemistry is growing rapidly [1,2,3,4]. Most of the biocatalysts
in current use, however, are limited to cofactor-independent enzymes such as hydrolases, which perform relatively
simple chemistry. In comparison, cofactor-dependent
www.current-opinion.com
enzymes, such as oxidoreductases and transferases, are
capable of performing much more complex chemistry and
catalyzing a large number of synthetically useful reactions. Cofactors are low molecular weight compounds that
are essential for enzymatic reactions. Certain cofactors
such as pyridoxal phosphate and biotin are tightly bound
to the enzymes and are essentially self-regenerating.
Others, such as pyridine nucleotides and nucleoside
triphosphates (NTPs), act as functional group transfer
agents and are cosubstrates that must be used in stoichiometric amounts [5]. As these cofactors are too expensive
to be used as stoichiometric agents for preparative applications, they must be regenerated in situ. Cofactor regeneration can also reduce the cost of synthesis by driving the
reaction to completion, simplifying product isolation,
and preventing the accumulation of inhibitory cofactor
by-products [3].
A practically useful cofactor regeneration method must
fulfill several requirements. The enzymes, reagents and
equipment required should be readily available, inexpensive, easily manipulated and stable under the operational
conditions. The energetics of the regeneration step
should be favorable both kinetically and thermodynamically. Any reagents or by-products of the regeneration step
should not interfere with product isolation and should be
compatible with the rest of the reaction system. Most
importantly, the ‘total turnover number’ (TTN) of the
cofactor should be high [5]. TTN is defined as the total
number of moles of product formed per mole of cofactor
during the course of a complete reaction [6]. This number
reflects several factors including the loss of cofactor due to
degradation or incorrect regiochemistry of regeneration,
the reaction time, and the catalyst turnover number (kcat).
As a rule of thumb, TTNs of 103 to 105 can be sufficient to
make a process economically viable depending on the
value of the reaction product.
In the past two decades, many chemical, enzymatic and
electrochemical methods have been investigated to
regenerate various cofactors [5,7]. Compared with enzymatic methods, chemical and electrochemical strategies
often lack the high selectivity required to achieve high
TTNs, and are frequently incompatible with the other
components of the enzymatic reactions. Thus, enzymatic
methods are presently preferred for cofactor regeneration.
As listed in Table 1, many enzymatic approaches have
been developed to effectively regenerate various cofactors, including adenosine 50 -triphosphate (ATP) regeneration in phosphoryl transfer reactions, NAD(P)þ or
NAD(P)H regeneration in oxidoreductions, acetyl CoA
regeneration in acyl transfer reactions, sugar nucleotide
Current Opinion in Biotechnology 2003, 14:583–589
584 Chemical biotechnology
Table 1
Common cofactors required for biotransformation and their representative in situ regeneration methods.
Cofactor
þ
NAD
NADH
NADPþ
NADPH
ATP
Sugar nucleotides
CoA
PAPS
S-Adenosyl methionine
Flavins
Pyridoxal phosphate
Biotin
Metal porphyrin complexes
Reaction type
Representative regeneration method
References
Removal of hydrogen
Addition of hydrogen
Removal of hydrogen
Addition of hydrogen
Phosphoryl transfer
Glycosyl transfer
Acyl transfer
Sulfuryl transfer
Methyl transfer
Oxygenation
Transamination
Carboxylation
Peroxidation, oxygenation
Glutamate dehydrogenase with a-ketoglutarate
Formate dehydrogenase with formate
Glutamate dehydrogenase with a-ketoglutarate
Glucose dehydrogenase with glucose
Acetate kinase with acetyl phosphate
Bacterial coupling
Phosphotransacetylase with acyl phosphate
Aryl sulfotransferase IV with p-nitrophenyl sulfate
No demonstrated method
Self-regeneration
Self-regeneration
Self-regeneration
Self-regeneration
[43]
[44]
[43]
[45]
[46]
[47]
[48]
[40]
[3]
[1]
[1]
[1]
[1]
Many flavin-dependent mono-oxygenases or di-oxygenases require additional NAD(P)H as an indirect reducing agent. A first example of
direct regeneration of a flavin-dependent mono-oxygenase was reported recently [41] (see also Update).
regeneration in glycosylations, and 3-phosphoadenosine50 -phosphosulfate (PAPS) regeneration in sulfuryl transfer reactions. Some of these have been successfully used
in large-scale chemical syntheses [5,7].
In this review, we will discuss new developments in
cofactor regeneration since 2000. We will focus on new
and improved approaches for regeneration of ATP, sugar
nucleotides and PAPS, and their applications in biocatalysis. We will not discuss the recent advances in pyridine
nucleotide or nicotinamide cofactor regeneration as this
topic has been reviewed recently [8].
ATP/NTP regeneration
NTPs are required in many synthetically useful enzymecatalyzed phosphoryl transfer reactions. ATP is most
frequently used as a phosphorylating agent and hence
has received more attention than other NTPs such as
uridine 50 -triphosphate (UTP) and cytidine 50 -triphosphate (CTP), which have been mainly used as sources
of nucleoside phosphates rather than suppliers of phosphate or energy [6]. Several enzyme and whole-cell based
methods have been developed to regenerate ATP from
adenosine 50 -diphosphate (ADP) [6,9]. Three enzymatic
methods are commonly employed: the use of phosphoenolpyruvate (PEP) as the phosphate donor in a coupled
reaction catalyzed by pyruvate kinase; acetylphosphate
coupled with acetate kinase; and polyphosphate coupled
with polyphosphate kinase (PPK). It should be noted that
because these three enzymes have broad substrate specificities, these ATP regeneration methods can also be
used to replenish other NTPs including guanosine 50 triphosphate (GTP), UTP and CTP from their corresponding nucleoside diphosphates [6,9].
Although the above-mentioned ATP regeneration methods are effective for enzyme-catalyzed chemical synthesis, they are not well-suited for cell-free protein
Current Opinion in Biotechnology 2003, 14:583–589
synthesis because of the high cost of substrates and
enzymes and, more importantly, because of the degradation of the phosphate-containing substrates by uncoupled
enzymes in the cell extracts. In addition, the accumulation of phosphates in the cell-free system will inhibit
long-term protein synthesis [10]. To address these limitations, a novel ATP regeneration approach was developed
for cell-free protein synthesis. This approach takes advantage of the endogenous enzymes present in cell extracts
and uses glycolytic intermediates such as pyruvate and
glucose-6-phosphate rather than PEP as energy sources.
It was found that with the addition of NADþ, certain
endogenous enzymes can convert pyruvate into acetylphosphate, which in turn is converted into acetate with
concurrent regeneration of ATP. Similarly, glucose-6phosphate can be first converted into pyruvate and then
acetate, with concurrent ATP regeneration occurring in
each step. As NADþ, pyruvate and glucose-6-phosphate
are much cheaper than PEP, and as the protein synthesis yield using these substrates is either comparable
to (for pyruvate/NADþ) or higher than (for glucose6-phosphate/NADþ) that using PEP, this new ATP
regeneration approach can significantly improve the economic efficiency of cell-free protein synthesis [10].
Another important recent advance in ATP regeneration is
the use of inorganic polyphosphate (poly(P)) to regenerate ATP from adenosine 50 -monophosphate (AMP)
[11,12,13]. An efficient method for regenerating ATP
from AMP is highly desirable, because many ATP-dependent enzymes produce AMP rather than ADP. Poly(P)
consists of linear polymers of orthophosphate containing
high-energy phospho anhydride linkages [14,15] and is
used as a phosphate donor because of its high stability and
low cost. Resnick and Zehnder [13] designed an ATP
regeneration system using poly(P) and the enzymes polyphosphate:AMP phosphotransferase (PAP) and adenylate
kinase (AdK) of Acinetobacter johnsonii. The system was
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Regeneration of cofactors for use in biocatalysis Zhao and van der Donk 585
successfully applied to synthesize glucose-6-phosphate
with hexokinase. In this process, hexokinase generates
ADP, which is converted by AdK into ATP and AMP. In
turn, the AMP is phosphorylated by PAP to ADP. The
main advantage of this approach lies in the use of relatively inexpensive AMP as the initial nucleotide and
poly(P) as the phosphoryl donor. Current drawbacks that
are likely to be addressed in future research concern the
low TTN (3–4) achieved in this preliminary study and
the lack of a cloned gene encoding PAP. In conjugation
with ATP-dependent light-emitting luciferase, this same
system was also adapted to detect AMP for hygiene monitoring and RNA quantification [16]. Shiba and coworkers
[11] developed a similar process in which PAP from
Myxococcus xanthus and a recombinant PPK from Escherichia coli were used together to regenerate ATP from AMP
and poly(P). This strategy was successfully used in combination with acetyl-CoA synthase to synthesize acetylCoA with the addition of inorganic pyrophosphatase
(PPase) to alleviate the inhibitory effect of PPi and to
provide an additional driving force (Figure 1). Compared
with the PAP–AdK system, this approach appears to
have several advantages, including a TTN for the ATP
cofactor of 40 and the ability to regenerate GTP from
GMP using the same system. PAP and PPK accept the low
cost phosphate donor poly(P), but unfortunately they are
both highly specific for their nucleotide substrate, AMP
and ADP, respectively [17]; hence, ATP regeneration from
AMP presently always requires two enzymes.
As with the ATP regeneration methods from AMP, a
novel enzymatic method using poly(P) as sole phosphorus
donor has been developed to regenerate CTP from CMP
[18]. In this strategy, the combined activity of PPK and
cytidine 50 -monophosphate (CMP) kinase from E. coli
converts CMP into CDP, while PPK by itself further
phosphorylates CDP. This regeneration system was
Figure 1
poly(P)n
poly(P)n–1
ADP
PPK
PAP
poly(P)n
poly(P)n–1
ATP
AMP + PPi
Acetate + CoA
PPase
2Pi
Acetyl-CoA
Acetyl-CoA synthase
The PAP-PPK ATP regeneration system from AMP coupled with the
acetyl-CoA synthetic reaction. PPase, inorganic pyrophosphatase.
(Figure adapted from [11] with permission.)
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coupled with cytidine 50 -monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase of Haemophilus
influenzae to synthesize CMP-NeuAc. It should be noted
that CMP-NeuAc can also be produced by a novel recombinant E. coli strain containing an acetylphosphate/
acetate kinase-based ATP regeneration system [19].
These two studies will be further discussed below.
Sugar nucleotide regeneration
Oligosaccharides and polysaccharides have vital roles in a
variety of biological processes including molecular recognition and cellular communication in bacterial and viral
infection, cancer and the immune response. On this basis,
they have been explored as potential pharmaceuticals
[20,21]. Among the numerous methods developed for
oligosaccharide synthesis, glycosyltransferase-mediated
methodology is recognized as one of the most practical
approaches for large-scale preparation, owing to the high
regioselectivity and stereoselectivity of these enzymes
[20]. However, glycosyltransferases require a sugar
nucleotide as donor substrate that must be generated
in situ for preparative applications by recycling of the
nucleotide moiety. Although many glycosyltransferases
are involved in the biosynthesis of oligosaccharides, only
nine sugar nucleotides are used in mammalian systems,
including uridine 50 -diphosphoglucose (UDP-Glc), UDPgalactose (UDP-Gal), UDP-glucuronic acid (UDPGlcUA), CMP-NeuAc, guanosine 50 -diphosphomannose
(GDP-Man), GDP-fucose (GDP-Fuc), UDP-N-acetylglucosamine (UDP-GlcNAc), uridine 50 -diphospho-a-Dxylose (ADP-Xyl) and UDP-N-acetylgalactosamine
(UDP-GalNAc) [22]. The enzymatic synthesis of these
compounds has been reviewed [23].
Recently, several new approaches have been developed to
regenerate sugar nucleotides including UDP-Gal, UDPGlc, UDP-GlcNAc, CMP-NeuAc and GDP-Fuc for preparative applications. One notable approach is bacterial
coupling, which uses a combination of batches of different
strains of whole cells. For instance Corynebacterium ammoniagenes, which efficiently produces UTP from orotic acid,
has been coupled with recombinant E. coli strains expressing the enzymes required for sugar nucleotide generation. Importantly, this strategy does not require enzyme
purification or the addition of nucleotides. The approach
has been successfully used for large-scale production
of sugar nucleotides including CMP-NeuAc (17 g l1),
UDP-GlcNAc (7.4 g l1) and GDP-Fuc (18.4 g l1)
[24,25,26]. As illustrated in Figure 2, CMP-NeuAc
can be obtained by the combination of C. ammoniagenes
and two E. coli strains expressing CTP synthetase (converting UTP to CTP) and CMP-NeuAc synthase (converting NeuAc and CTP to CMP-NeuAc), respectively
[24]. Further coupling of the CMP-NeuAc or GDP-Fuc
regeneration systems with recombinant E. coli strain(s)
over-expressing desired glycosyltransferase(s) led to largescale production of several important oligosaccharides
Current Opinion in Biotechnology 2003, 14:583–589
586 Chemical biotechnology
Figure 2
Recombinant E. coli #2
NeuAc
neuA
Lac
CMP-NeuAc
CTP
Recombinant
E. coli #1
pyrG
Recombinant E. coli #3
α-2,3-Sialyltransferase
CTP
UTP
CMP
CDP
3′ -Sialyllactose
UDP
Orotic acid
UMP
C. ammoniagenes
Current Opinion in Biotechnology
The CMP-NeuAc regeneration system using bacterial coupling. The system consists of C. ammoniagenes, which converts orotic acid into UTP, and
two recombinant E. coli strains expressing the CTP synthetase gene (pyrG) from E. coli K12 and the CMP-NeuAc synthetase gene (neuA) from
E. coli K1, respectively. Further coupling of a recombinant E. coli strain overexpressing the a-(2!3)-sialyltransferase gene from Neisseria gonorrhoeae
led to the large-scale production of 30 -sialyllactose using NeuAc, orotic acid and lactose (Lac) as substrates.
including globotriose, 3’-sialyllactose and Lewis X
[24,26]. The same strategy was also used to produce
the sialyl-Tn epitope of the tumor-associated carbohydrate antigen [27] and NeuAc [28].
Owing to the low cost of enzymes and starting materials,
bacterial coupling is a cost-effective method for sugar
nucleotide regeneration. However, because multiple
strains are used simultaneously, the transport of reaction
intermediates between strains and the maintenance of
cell growth of different strains may be problematic for
certain applications. To overcome these limitations, a
new approach using a single recombinant bacterial strain
(‘superbug’) containing all the necessary enzymes for
sugar nucleotide regeneration and oligosaccharide synthesis has been recently demonstrated [19,29,30]. Kim
and coworkers [19] developed a CMP-NeuAc regeneration method by co-expressing CMP kinase, sialic acid
aldolase and CMP-NeuAc synthetase in a single E. coli
strain. In related work, Wang and coworkers [29] introduced an artificial biosynthetic pathway consisting of the
genes encoding a galactosyltransferase, a UDP-galactose
4-epimerase from E. coli K-12 ( galE), and a sucrose
synthase from Anabaena sp. PCC 7119 (susA) into a single
E. coli strain. By choosing different galactosyltransferases,
these recombinant strains have been used for large-scale
production of the biomedically important trisaccharides
Gala1,4Lac (globotriose) and Gala1,3Lac (a-Gal epitope)
with the concurrent regeneration of UDP-Gal in a twostep process. The first step entailed the fermentation of
the engineered recombinant strain and the second step
Current Opinion in Biotechnology 2003, 14:583–589
involved the use of permeabilized harvested cells as
whole-cell biocatalysts for oligosaccharide synthesis. In
the same manner, an artificial gene cluster encoding all
five enzymes in the biosynthetic pathway of Gala1,3Lac
was transferred into an E. coli strain to produce the a-Gal
epitope [30].
Compared with the above-mentioned whole-cell based
approaches for sugar nucleotide regeneration and oligosaccharide synthesis, enzymatic methods can offer more
flexibility in certain cases. In particular, greater productivity and a simpler process for product isolation can be
achieved when immobilized enzymes are used. Wang and
coworkers [31,32] developed a new enzymatic method
for cost-effective UDP-Gal regeneration by immobilizing
seven enzymes involved in the biosynthesis of UDP-Gal
onto nickel agarose beads. The genes encoding all of the
enzymes were cloned and expressed individually, and the
corresponding enzymes were immobilized through their
hexahistidine tags to the beads [33]. The formation of
UDP-Gal was achieved using a combination of stoichiometric amounts of UMP and galactose with catalytic
quantities of ATP and glucose-1-phosphate, resulting
in a 50% yield based on UMP. In a subsequent study,
the methodology was used to prepare several oligosaccharides including globotriose and the a-Gal epitope
[32]. Although this approach has many advantages, the
authors point out that, to date, bacterial coupling is still
more cost effective. Ishige and coworkers [18] described a
new efficient enzymatic method for CMP-NeuAc regeneration based on purified enzymes. In this method, the
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Regeneration of cofactors for use in biocatalysis Zhao and van der Donk 587
gene encoding a novel CMP-NeuAc synthetase of H.
influenzae was cloned and the corresponding enzyme
was coupled with an enzymatic CTP regeneration system
discussed in the previous section.
aryl sulfotransferase IV with p-nitrophenyl sulfate as the
sulfate donor. This method proved to be most practical in
the preparative synthesis of carbohydrate sulfates [40].
Conclusions
In addition to the nine sugar nucleotides involved in
mammalian glycobiology, the dTDP- and UDP-activated
deoxyhexoses involved in secondary metabolism in bacteria and plants [34] have received much attention in
recent years for their potential application in reprogramming of the structures of antibiotics that are decorated
with carbohydrates [35]. Many of the glycosyltransferase
genes have been cloned and chemoenzymatic syntheses
of various pyrimidine diphosphosugars with sugar nucleotide regeneration have been reported [36–38].
PAPS regeneration
The cofactor PAPS is the universal sulfate donor in biological systems and is required in the sulfotransferasecatalyzed synthesis of many biologically or therapeutically
important sulfated carbohydrates and glycopeptides.
The high cost and instability of PAPS and the production inhibition problem caused by 30 -phosphoadenosine
50 -sulfate requires in situ regeneration of PAPS for
preparative applications.
The first enzymatic PAPS regeneration method was
based on a complex multi-enzyme system consisting of
ATP sulfurylase, adenosine-50 -phosphosulfate (APS)
kinase, 30 -nucleotidase, myokinase and pyruvate kinase
[39]. In this process, which mimics the natural metabolic
cycle, the product generated after sulfate transfer is first
converted to ATP using the latter three enzymes; ATP is
subsequently transformed to PAPS by ATP sulfurylase
and APS kinase. Recently, a much simpler, one-enzyme
PAPS regeneration method has been developed
(Figure 3) [40]. This method uses a recombinant rat liver
Figure 3
Substrate
Sulfated product
NH2
NH2
PAPS
N
N
N
Target
sulfotransferase
N
O
–
O
O S
O–
N
O
O P O
O P O
N
N
N
O
O–
O
O
O–
OH
O P O–
O
OH
O P O–
O–
Aryl
sulfotransferase
O–
O–
O2N
OH
O 2N
O
S
O
O
p-Nitrophenyl sulfate
Much of the recent progress in cofactor regeneration has
focused on nicotinamide cofactors, ATP, sugar nucleotides
and PAPS. In the case of ATP regeneration, the use of
inexpensive poly(P) as sole phosphorus donor coupled with
PAP and PPK/AdK enables the regeneration of ATP from
AMP. Because PPK catalyzes the NTP regeneration reaction using poly(P) only, it might also be worthwhile engineering or discovering PPK with novel nucleotide
monophosphate (NMP) specificity for the construction
of an NTP regeneration method from NMP. In addition,
a novel ATP regeneration method using glycolytic intermediates such as pyruvate and glucose-6-phosphate rather
than PEP as energy sources was developed for the costeffective synthesis of proteins in a cell-free system. Among
the several newly developed sugar nucleotide regeneration
methods, whole-cell approaches using bacterial coupling or
a single genetically engineered bacterial strain represent
the most efficient and cost-effective routes for production
of sugar nucleotides and pharmaceutically important oligosaccharides. With the discovery of more bacterial glycosyltransferases from genomics- and proteomics-related
research and the development of better protein expression
systems, the whole-cell approaches will receive much
attention in glycobiotechnology in the near future.
Furthermore, the development of a novel one-enzyme
based PAPS regeneration method enables the cost-effective large-scale production of sulfated carbohydrates. In
conjugation with the newly developed sugar nucleotide
regeneration methods, this PAPS regeneration system is
expected to facilitate the production of more complex
carbohydrates of biological and therapeutic importance.
Although much progress has been made in the past two
decades, further improvement of the existing regeneration methods for nicotinamide cofactors, ATP/NTP,
sugar nucleotides and PAPS will be required for their
expected increased application in biocatalysis. Along this
line, modern protein engineering and metabolic engineering techniques will be increasingly used, taking
advantage of the advances in genomics, proteomics and
bioinformatics. Future efforts should also focus on the
development of new methods for cost-effective regeneration of S-adenosyl methionine (SAM), which currently is
an unsolved problem on a preparative scale. It is expected
that, with the development of inexpensive and efficient in
situ regeneration methods for various cofactors, the applications of biocatalysis in the pharmaceutical and chemical
industries will be greatly expanded in the coming years.
Current Opinion in Biotechnology
Update
A novel PAPS regeneration method comprising a recombinant aryl
sulfotransferase and p-nitrophenyl sulfate.
www.current-opinion.com
Hollmann and coworkers [41] recently developed a
novel chemo-enzymatic approach to directly regenerate
Current Opinion in Biotechnology 2003, 14:583–589
588 Chemical biotechnology
flavin-dependent styrene monooxygenase (StyA) for the
first time by means of an organometallic complex
[CpRh(bpy)(H2O)]2þ). This organometallic complex
can catalyze the transhydrogenation reaction between
formate and isoalloxazine-based cofactors such as FAD
and FMN, and thereby substitutes for the native reductase (StyB), the nicotinamide coenzyme (NAD), and an
enzymatic NADH regeneration system such as formate
dehydrogenase in StyA-catalyzed epoxidation reactions.
Similarly, Wagenknecht and coworkers [42] developed a
simple system based on ruthenium (II) and rhodium (III)
organometallic complexes for the direct coupling of
hydrogen to nicotinamide cofactor regeneration under
conditions appropriate for in situ coupling with enzymatic
reductions. The use of hydrogen as a reducing agent is
advantageous compared with other reducing agents;
however, the TTNs of the catalysts are very low.
Bommarius and coworkers [43] demonstrated for the first
time that NADH oxidase from Lactobacillus sanfranciscensis can be used to regenerate both NADþ from NADH
and NADPþ from NADPH with concomitant reduction
of O2 to innocuous H2O. Currently, the turnover number
of the enzyme is still low, but is likely to be amenable to
improvement.
Acknowledgements
We thank Biotechnology Research and Development Consortium (BRDC)
for supporting our work on developing a phosphite dehydrogenase-based
nicotinamide cofactor regeneration system for reductive biocatalysis.
References and recommended reading
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of special interest
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