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Biotechnology Advances 25 (2007) 369 – 384

Research review paper

Cofactor regeneration for sustainable enzymatic biosynthesis
Wenfang Liu a , Ping Wang b,⁎
a
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, China
b
Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN 55108, United States
Received 11 January 2007; received in revised form 3 March 2007; accepted 12 March 2007
Available online 23 March 2007

Abstract

Oxidoreductases are attractive catalysts for biosynthesis of chiral compounds and polymers, construction of biosensors, and
degradation of environmental pollutants. Their practical applications, however, can be quite challenging since they often require
cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These
cofactors are generally expensive. Efficient regeneration of cofactors is therefore critical to the economic viability of industrial-
scale biotransformations using oxidoreductases. The chemistry of cofactor regeneration is well known nowadays. The challenge is
mostly regarding how to achieve the regeneration with immobilized enzyme systems which are preferred for industrial processes to
facilitate the recovery and continuous use of the catalysts. This has become a great hurdle for the industrialization of many
promising enzymatic processes, and as a result, most of the biotransformations involving cofactors have been traditionally
performed with living cells in industry. Accompanying the rapidly growing interest in industrial biotechnology, immobilized
enzyme biocatalyst systems with cofactor regeneration have been the focus for many studies reported since the late 1990s. The
current paper reviews the methods of cofactor retention for development of sustainable and regenerative biocatalysts as revealed in
these recent studies, with the intent to complement other reviewing articles that are mostly regeneration chemistry-oriented. We
classify in this paper the methods of sustainable cofactor regeneration into two categories, namely membrane entrapment and solid-
attachment of cofactors.
© 2007 Elsevier Inc. All rights reserved.

Keywords: Oxidoreductases; Enzyme immobilization; Cofactor regeneration; Biocatalysis; Biosynthesis; Industrial biotechnology

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
2. Membrane entrapment of free cofactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
2.1. Native cofactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
2.2. Chemically modified cofactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
2.2.1. Chemical modification of cofactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
2.2.2. Retention of chemically modified cofactors by using UF membranes . . . . . . . . . . . . . . . . . 375

⁎ Corresponding author. Tel.: +1 612 624 4792.

E-mail address: ping@umn.edu (P. Wang).

0734-9750/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2007.03.002
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370 W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384

2.2.3. Retention of chemically modified cofactors by using microcapsules . . . . . . . . . . . . . . . . . 375

2.2.4. Retention of chemically modified cofactors on electrodes . . . . . . . . . . . . . . . . . . . . . . 376
3. Solid-phase cofactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
3.1. Physically immobilized cofactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
3.1.1. Physical attachment of cofactor for organic synthesis . . . . . . . . . . . . . . . . . . . . . . . . 376
3.1.2. Physical attachment of cofactors on electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
3.2. Chemical incorporation of cofactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

1. Introduction is particularly preferred for industrial processes due to

Oxidoreductases represent about one quarter of the
known enzymes (Kula and Kragl, 2000). Awide spectrum
of applications have been explored so far for this group of
enzymes, including synthesis of chiral compounds, such
as chiral alcohols, aldehydes and acids; preparation and
modification of polymers, especially biodegradable or
biocompatible polymers; biosensors for a variety of
analytical and clinical applications; and degradation of
organic pollutants (Sheldon and Stephen, 1983; Hummel,
1999). Due to their high efficiency and specificity, these
enzymes are particularly attractive for biosynthesis.
Oxidoreductases generally require a non-protein chemical
group, a cofactor, to catalyze reactions. Although certain
oxidoreductases possess prosthetic groups to facilitate
reactions, the majority of the enzymes explored for
biosynthesis needed to interact with cofactors that are not
permanently tethered to the enzymes. The most widely
involved cofactors are several organic compounds, which
are also often referred to as coenzymes, such as NAD(H),
NADP(H) and ATP. In particular, NAD(H) and NADP(H)
have been examined extensively in recent years for
chemical processing applications.
Unlike enzymes, cofactors act as stoichiometric agents
in biotransformation reactions and undergo chemical
reactions with substrates. Often they are much more ex-
pensive than the desired products. Accordingly, efficient
regeneration and reuse of the cofactors are essential to
large-scale synthetic applications (Chenault et al., 1988;
Wichmann and Vasic-Racki, 2005). A total turnover
number (TTN) of the cofactors, defined as mol product
produced/mol cofactor applied, in the order of hundreds up
to thousands is usually desired to make the biocatalytic
processes economically viable (Chenault et al., 1988).
Methods including chemical, electrochemical, pho-
tochemical, microbial and enzymatic reactions have all
been developed for cofactor regeneration (Chenault and
Whitesides, 1987). Among others, enzymatic approach
its high selectivity and efficiency. It also affords the
feasibility of coupling more than one valuable chemical
production routes. There are two different ways to
achieve enzymatic regeneration (Fig. 1). One is through
the use of substrate-coupled reaction systems, in which
one enzyme that uses both the reduced and oxidized
forms of a cofactor is applied to catalyze both the
desired synthesis of the product from one substrate and
the cofactor regeneration reaction with a second
substrate. One example for that is the alcohol dehydro-
genase (ADH)-catalyzed organic synthesis reaction.
Acetophenone was reduced enantioselectively into (S)-
1-phenylethanol by an NADPH-dependent ADH from
Thermoanaerobacter sp., and the conversion of acet-
ophenone could reach 98% when 2-propanol was used
as the secondary substrate to drive the regeneration of
NADPH catalyzed by the same ADH in a batch reactor
(Findrik et al., 2005). Since the same enzyme is required
to catalyze two separated reactions simultaneously, it is
usually difficult to achieve thermodynamically-favorite
reaction conditions for both reactions in the same
reaction medium. The other way is through the use of a
second enzyme to catalyze the cofactor regeneration
reaction. The use of a second enzyme, which has been
adapted for the majority of cofactor regeneration pro-
cesses, usually affords broader options of substrates for
the cofactor regeneration reaction, and thus makes it
much easier to achieve large thermodynamic driving
Fig. 1. Enzymatic regeneration of cofactors (for substrate-coupled
regeneration, the two enzymes are the same, E1 = E2; for enzyme-
coupled regeneration, E1 and E2 represent two different enzymes).
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W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384 371

Fig. 2. Cofactor regeneration with electrochemical reactions. (a) Electro-enzymatic cofactor regeneration: the cofactor is regenerated through an
enzyme-catalyzed reaction; redox reactions on electrodes regenerate the second substrate, the mediator; Medred and Medox represent the reduced and
oxidized forms of mediator, respectively. (b) Electrochemical cofactor regeneration: the cofactor undergoes redox reactions directly on the electrode.

forces for both reactions. In either way, it is desirable to
make value-added products from the second substrates
applied for cofactor regeneration; otherwise, the second
substrates have to be either very cheap or can be
regenerated easily for reuse. Electrochemical reactions
are usually introduced for the latter case, i.e., the second
substrate applied for cofactor regeneration is regener-
ated electrochemically (Scheper et al., 1987; Montagné
and Marty, 1995; Nakamura et al., 1996; Leca and
Marty, 1997a,b; Noguer and Marty, 1997; Maines et al.,
2000; Leonida, 2001). For such a purpose, the second
substrates, usually referred to as mediators, are required
to possess both good electrochemical reactivity on
electrodes and good reactivity with the enzyme and
cofactor (Fig. 2(a)).
Cofactors can be regenerated directly on the surface
of electrodes without the involvement of mediators
and enzymes (Fig. 2(b)). We may define this method
as electrochemical regeneration of cofactors. In the case
of mediator-assisted regeneration as discussed above,
the cofactor is regenerated by an enzyme-catalyzed
reaction, while the mediator is regenerated on electro-
des. The mediators are usually much more active than
cofactors for electrochemical reactions on electrodes,
and thus provide the possibility for higher overall
reaction rates.
As mentioned earlier, efficient cofactor regeneration
is highly desired in biosynthesis. A high TTN is difficult
to achieve, however, if the products accumulate in the
reaction media and build thermodynamic resistance

Fig. 3. Typical configurations for sustainable cofactor retention in continuous-flow bioreactors.

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372 W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384
Fig. 4. Membrane entrapment of native cofactors. (a) Retention via size exclusion using uncharged membranes; (b) retention with charged
membranes; (c) retention with uncharged UF membranes with the aid of polyelectrolytes.
against the forward reactions. Accordingly, continuous-
flow reactors (Fig. 3) that allow continuous feeding of
substrates and removal of products are always preferred
in large-scale synthetic applications. While continuous-
flow reactors have been applied widely and successfully
with enzymes that do not require cofactors, it has been a
daunting task with cofactor-associated reactions as the
cofactors tend to leave the reactors along with the
substrates and products. How to achieve the immobili-
zation of the cofactors while maintaining their func-
tionality has proved to be considerably challenging.
Earlier methods for cofactor immobilization have been
reviewed previously by Lowe (Lowe, 1978), while the
broader biochemical potentials of cofactors have been
reviewed more recently (Chenault and Whitesides,
1987; Peters, 1999; Zayats et al., 2000). The current
review focuses on recent advances in developing
immobilized cofactors for sustainable biocatalysis with
cofactor regeneration. Herein we classify the methods
for sustainable cofactor regeneration for continuous-
flow processes into two categories: (1) membrane
entrapment of dissolved free cofactors, which could be
either native cofactors or chemically modified, with
various forms of membranes including nano-filtration
(NF) membranes, ultrafiltration (UF) membranes,
dialysis membranes, microcapsules, etc.; and (2) solid-
attachment of cofactors, including both physical and
chemical incorporation of cofactors into insoluble solid
supports.
Most of the works reviewed are publications in 1990s
and later, with several papers on modification chemistry
of cofactors dating back to 1970s. Since the focus is on
the retention of cofactors in bioreactors, many publica-
tions concerning the chemical aspects of cofactor
regeneration reactions are not included. All the methods
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reviewed here are those published in literature and
examined through lab-scale experiments. Little is
known regarding which methods have been practiced
in industry. In fact, according to recent reviews of
industrial biocatalysis (Liese et al., 2000), only several
enzymatic processes involving cofactor regeneration
have been practiced in industry. These include the
lactate dehydrogenase (LDH)/NADH/formate dehydro-
genase (FDH), alcohol dehydrogenase (ADH)/NADH/
FDH, ADH/NADH/glucose dehydrogenase (GDH),
ADH/NADPH/GDH, and leucine dehydrogenase
(LeuDH)/NADH/FDH systems (Liese et al., 2000).
While the chemistry for these processes was reported,
little is known regarding how the cofactors were
handled in the industrial bioreactors.
2. Membrane entrapment of free cofactors
2.1. Native cofactors
Compared to enzymes, cofactors are small mole-
cules. That makes it difficult to choose an appropriate
filter membrane to retain the cofactors while allowing
the products and substrates to pass through (Fig. 4). In
the study for production of l-glutamate and l-carnitine
catalyzed by glutamate dehydrogenase (GLDH) and l-
carnitine dehydrogenase (CDH), Lin and coworkers
used a NF membrane, UTC-20, to partially retain NAD+
or NADP in the reactor (Lin et al., 1997, 1999). GDH
was applied for the regeneration of NAD+ and its TTN
was reported to be up to 10,000. The cofactors could still
penetrate through the membrane and a certain amount of
cofactors must be continuously amended along with the
substrates for a continuous production. Membranes with
smaller pores will improve the retention of cofactors;
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W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384
however, that will lead to a reduced flow rate of
substrates (Obón et al., 1998).
How to prevent the loss of cofactors from reactors
while maintaining high substrate flow rates has been a
long-standing challenge. Over the years, different
methodologies have been conceived and tested. One
approach is to employ charged membranes (Fig. 4(b)),
which help to retain cofactors via electrostatic repulsion
instead of size exclusion (Kitpreechavanich et al., 1985;
Howaldt et al., 1990; Ikemi et al., 1990a; Ikemi et al.,
1990b; Kulbe et al., 1990; Röthig et al., 1990; Nidetzky
et al., 1994; Nidetzky et al., 1996). For example, a
negatively charged NF membrane, NTR 7410, was used
for reactions involving NADP(H), which possesses
negatively charged phosphate moieties (Ikemi et al.,
1990b). The conversion of fructose into sorbitol coupled
with GDH-catalyzed cofactor regeneration was operated
continuously for over 800 h, and the TTN of NADPH
was 106,000. In a continuous process for the synthesis
of sulcatol using ADH, the cofactor NADP(H) was
retained by a charged UF membrane and regenerated by
oxidizing isopropanol to acetone (Röthig et al., 1990). It
was claimed that the overall efficiency of NADP(H)
with charged UF membrane was even better than that for
poly(ethylene glycol) (PEG)-coupled NADP(H) with an
uncharged UF membrane. The application of a sulfo-
nated polysulfone membrane for the retention of NADP
(H) and NAD(H) was also investigated (Kitpreechava-
nich et al., 1985; Howaldt et al., 1990). An alternative
approach is to use polyelectrolyte like polyethylenei-
mine (PEI) with uncharged UF membranes (Fig. 4(c)).
The polyelectrolyte can complex with the charged
cofactors and enzymes and thus help to maintain them
in reactors and allows the use of filter membranes with
larger pores. This approach was demonstrated for the
production of l-lactate, gluconate and glutamate (Obón
et al., 1996, 1998). Although better retention of cofactor
can be generally expected, the effectiveness of the
electrostatic repulsion method is also subject to the
effects of several other factors, including the charges
carried by the products, substrates, salts, and other
chemicals in the reaction media (Howaldt et al., 1990;
Kulbe et al., 1990).
The volume and time productivity of bioreactors with
membrane-retained cofactors is largely determined by
the effective area of the membranes. Microcapsules,
which provide much more membrane area per unit
volume than fixed membranes, were therefore devel-
oped for the retention of cofactors. Chang and cow-
orkers developed microcapsules using lipid–polymer
complex membranes for capsulation of cofactors in the
early 1980s (Yu and Chang, 1981a,b, 1982; Chang et al.,
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373
1982; Ilan and Chang, 1986). By adjusting the ratio of
cholesterol to lecithin, they showed that such complex
membranes could afford very low permeability to lipid-
insoluble substances including cofactors, but at the same
time allow lipid-soluble substrates such as ammonia to
penetrate very freely. In one example, lipid–polyamide
membrane capsulated NADH, urease, GLDH and ADH
were applied for the production of amino acids from
urea or ammonia. NADH was regenerated by ADH in
the presence of ethanol and a maximum TTN of 40 was
achieved for NADH in 3 h (Yu and Chang, 1982).
Similarly, a liquid surfactant membrane was applied to
capsulate NADH-mediated reaction systems including
leucine dehydrogenase (LeuDH)–FDH and ADH–FDH
(Scheper et al., 1987; Orlich and Schomäcker, 1999;
Orlich et al., 2000). A detailed review on these works
was presented by Orlich and Schomäcker (2001).
Membrane entrapment has been applied widely for the
preparation of enzyme-based electrodes. Most of these
efforts were made for the development of biosensors or
biofuel cells, for which the lifetime and reliability of elec-
trodes are mostly concerned while high TTN of cofactors is
usually not a focus. Many reviews on biosensors using
enzyme-based electrodes have been published (for exam-
ple, see Gilmartin and Hart (1995) for carbon electrode
biosensors, and Cosnier (2003) and Vidal et al. (2003) for
more recent publications). Several examples from recent
publications are provided in the following to demonstrate
the methodologies of cofactor retention on electrodes,
which can be potentially applied for electrochemical
regeneration of cofactors for biosynthetic purposes.
A size-exclusion cellulose acetate (CA) membrane was
applied to contain lactate dehydrogenase (LDH) and its
cofactor NAD+ to the surface of a Meldola's blue (acting
as a mediator)-modified carbon electrode for the construc-
tion of a disposable amperometric biosensor for lactic acid.
By adjusting the composition of CA membrane, the
leaching of cofactor and other biomolecules could be
minimized (Sprules et al., 1995). Polylysine was also
applied for the capsulation of NAD+, GDH and mediator
on the electrode for use as biosensors (Mano and Kuhn,
2005). Similarly, an amperometric biosensor for alcohols
was obtained by coating a carbon electrode with a solution
containing ADH, NAD+ and poly(ester-sulfonic acid).
After drying, a polymeric membrane layer was applied to
cover the enzyme/cofactor layer. Ruthenium particles
dispersed on the electrode provided an efficient electro-
catalytic activity for the regeneration of NAD+ (Wang
et al., 1995). In order to improve the biocompatibility of
electrodes and improve their performance, a double-
membrane technique was developed to attach enzyme and
cofactor onto the electrode. A layer of poly(vinyl chloride)
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374 W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384
(PVC) membrane was first cast on a platinum electrode.
Native NAD+ and ADH were then deposited on the PVC
membrane, followed by covering with a cellulose triacetate
(CTA) membrane. It was reported that such electrodes had
good retention of NAD+, and demonstrated very good
sensitivity and operational stability for 30 times of mea-
surements within 4 weeks (Gotoh and Karube, 1994). In
the development of a malate biosensor, cofactor NAD+
and enzymes including malate dehydrogenase (MDH) and
diaphorase (DI) were first adsorbed on a high protein-
binding nitrocellulose membrane, which was subsequently
covered by a second polymeric membrane. It was found
that both a Tween-80 modified CA (5%CA + 5% Tween-
80) membrane and an un-plasticised spin-coated PVC/
polycarbonate (PC) resin membrane were effective to
retain cofactor and mediator while provide good perme-
ability to malate (Maines et al., 2000).
2.2. Chemically modified cofactors
2.2.1. Chemical modification of cofactors
Increasing the size of the cofactors would allow the
use of filters with larger pores and thus reduce the mass
transfer resistance of substrates/products. This can be
achieved by attaching chemical groups, such as
polymers, onto cofactors. The modifying groups should
be generally hydrophilic to maintain the aqueous
solubility of the cofactors, and should have appropriate
size to balance the reactivity and permeability of
cofactors. Common water-soluble macromolecular
modifiers that have been applied include PEI (Zappelli
et al., 1975, 1976, 1977; Riva et al., 1986), dextran
(Larsson and Mosbach, 1974; Grunwald and Chang,
1979, 1981, 1988a; Adachi et al., 1984; Gu and Chang,
1990a,b,c) and PEG (Furukawa et al., 1980; Bückmann
et al., 1981; Okuda et al., 1985; Riva et al., 1986;
Ottolina et al., 1990). Polylysine (Wykes et al., 1975;
Zappelli et al., 1975, 1976), poly(acrylic acid) (PAA)
Fig. 5. Typical chemical modification routes of cofactors. (a) Multi-step; (b) single-step.
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(Zappelli et al., 1977) and other soluble copolymers
(Fuller et al., 1980) were also examined. Compared to
PEI- and PAA-modified cofactors, PEG-NAD+ showed
better results with several dehydrogenases (Riva et al.,
1986). PEG-NADH also showed higher efficiency than
dextran-NADH for biosensor applications (Noguer and
Marty, 1997).
In some cases, the enzymes such as ADH that require
cofactors can serve as the modifiers for cofactors.
Among others, the complexes of ADH–NAD+ (Mån-
sson et al., 1978; Goulas, 1987; Sekhar and Plapp, 1988;
Vanhommerig et al., 1996; LeBrun and Plapp, 1999;
Leskovac et al., 2003) and LDH–NAD+ (Gacesa and
Venn, 1979; Schafer et al., 1986) were the most
extensively studied enzyme–cofactor conjugates. It
was reported that the reaction rate of a covalently linked
GDH–PEG-NAD+ conjugate was even higher than that
observed for native enzyme and native NAD+ (Naka-
mura et al., 1986). A similar approach was adopted for
MDH-catalyzed reactions (Eguchi et al., 1986). Genetic
engineering technique has also been applied for the
production of enzyme–cofactor conjugates. The pro-
duction of NAD+ tethered to specific sites of GDH from
Bacillus subtilis using site-directed mutagenesis by a
disulfide bridge has been reported (Månsson et al.,
1991).
The attachment of chemical groups can be achieved
by using spacers between the cofactor and modifier
(Srere et al., 1973). Zappelli et al. prepared a NAD+
analogue carrying a ω-carboxyalkyl side-chain (2-
hydroxy-3-carboxypropylamino) at exocyclic adenine
amino group, and then coupled it to PEI or polylysine
via the carbodiimide group (Zappelli et al., 1975). These
macromolecular NAD+ derivatives showed 25–60%
efficiency of free NAD+ with LDH, while only 2–7%
with yeast ADH (YADH) and alanine dehydrogenase
(AlaDH). When carboxyalkyl group was introduced at
the adenine C-8 position, PEI-NAD+ showed 47%
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W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384
efficiency with YADH while only 3% with LDH, and
polylysine-NAD+ showed 5–6% efficiency with both
enzymes (Zappelli et al., 1976). Riva et al. had done a
systematic investigation on the influence of coupling
site on the properties of cofactors when they were linked
to PEG, PEI or PAA (Riva et al., 1986). The results
showed that the N6 position of the adenine ring was the
optimum site, giving the most satisfactory results for
many dehydrogenases. In addition, NAD+ pre-modified
with succinyl group before coupling with PEI was
reported to retain better activity (Wykes et al., 1975).
The conjugation reaction of NADH with alkyl groups
may include three steps: (1) alkylation of NAD+ at N-1 of
adenine ring; (2) reduction of the nicotinamide moiety
with dithionite; and (3) Dimroth rearrangement of alkyl
linkage from the N-1 to the C-6 amino position (Fig. 5(a)).
Following this procedure, the preparation of PEG-N6-(2-
aminoethyl)-NADH and dextran-N 6 -(2-aminoethyl)-
NADH has been reported (Bückmann et al., 1981). By
incubating acrylic copolymer containing epoxy group
with NAD+ at pH 10, a one-step cofactor–polymer
coupling was also achieved (Fig. 5(b)) (Fuller et al.,
1980).
2.2.2. Retention of chemically modified cofactors by
using UF membranes
Chemically modified and thus physically larger
cofactors generally retain better in membrane reactors
as compared to native cofactors. Succinyl-NAD +
coupled to polylysine was applied for a membrane
reactor along with ADH and LDH. Continuous
production of lactate was performed, and the half-life
of the system was found to be 10 days (Yamazaki et al.,
1976). Loss of activity may come from either enzyme
deactivation or leakage of enzymes and cofactor. By
coupling N6-(2-carboxylethyl)-NAD+ to monoamino-
PEG (Mw 3000–3700) with water-soluble carbodii-
mide, PEG-NAD+ showed activity that was up to 77%
of native NAD+, depending on the dehydrogenases
applied. PEG (3000–3700)-NAD+ could keep active for
200 h when it was applied for lactate production with
enzymes LDH and YADH in a reactor with an UF
membrane (Furukawa et al., 1980). It was also reported
recently that PEG (10,000)-NAD+ was applied for
continuous synthesis of l-tert-leucine in a small-scale
UF-membrane reactor. The regeneration of the modified
cofactor was catalyzed by FDH, and a TTN of 125,000
was achieved (Filho et al., 2003). Dextran-modified
cofactors also showed promising results. Larsson and
Mosbach prepared dextran-NAD+ by coupling N6-[N-
(6-aminohexyl)-acetamide]-NAD+ with cyanogen bro-
mide activated dextran. Dextran-NAD+ remained about
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375
15% of the cofactor's original activity with either
YADH or LDH (Larsson and Mosbach, 1974). Dextran-
NAD+ had also been applied for continuous production
of alanine from pyruvate in an UF-membrane reactor.
The modified cofactor was repeatedly used for 90 times
with regeneration catalyzed by lactose dehydrogenase
(Davies and Mosbach, 1974). NAD+ coupled with an
enzyme, GDH from B. subtilis, was applied for a hollow
fiber–membrane reactor with LDH as a regenerating
enzyme for continuous production of l-lactate and
gluconic acid (Månsson et al., 1991). A TTN of
135,000 was achieved for the cofactor within the first
2.5 days.
2.2.3. Retention of chemically modified cofactors by
using microcapsules
Chang and coworkers conducted a series of studies
for the encapsulation of polymer-attached cofactors,
especially NAD+, with native enzymes entrapped with
ultra thin semi-permeable membranes (Campbell and
Chang, 1976; Grunwald and Chang, 1979, 1981;
Chang, 1985; 1987; Wahl and Chang, 1987; Gu and
Chang, 1988a,b, 1990a,b,c). For examples, microcap-
sules of cellulose nitrate membranes have been prepared
by using ether as the solvent and Tween-20 as an oil/
water emulsifier; while polyamide membrane micro-
capsules have prepared by interfacial polymerization of
terephthaloyl and diamine-PEI (Chang, 1985, 1987;
Chang and Prakash, 2001). In their work, dextran seems
to be the most popularly employed modifier for
cofactors (Grunwald and Chang, 1979; Gu and Chang,
1988a, 1990a,b,c), while PEI (Campbell and Chang,
1976), PEG (Stengelin and Patel, 2000), albumin and
hemoglobin (Wahl and Chang, 1987) were also used.
Among the several multi-enzyme systems that have
been encapsulated, YADH/MDH/polymer-NAD +
(Campbell and Chang, 1976; Grunwald and Chang,
1979; Wahl and Chang, 1987) and urease/LeuDH/
dextran-NAD+ (Gu and Chang, 1990a,b,c) systems have
been studied extensively. Following a similar strategy,
urease/GLDH/YADH/dextran-NAD+ (Gu and Chang,
1988a) and phenylalanine dehydrogenases(PheDH)/
FDH/PEG-NADH (Stengelin and Patel, 2000) systems
were also examined.
Unfortunately, some encapsulated multi-enzyme
systems did not show good stability. Cellulose nitrate-
capsulated YADH, MDH and dextran-NAD+ only retain
31% of its original activity after being stored for 7 days
(Grunwald and Chang, 1979). Interestingly, it was found
that the addition of protein (typically, 10 g/dL purified
hemoglobin) in the capsules could stabilize the enzyme
systems. It was believed that the added protein provided
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376 W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384
an intracellular environment that is comparable to red
blood cells (Chang, 1985). Addition of 10% PEI also
showed a certain stabilizing effect (Chang, 1987).
Further stabilization could be obtained by cross-linking
with glutaraldehyde (GA) (Chang, 1971, 1985, 1987;
Grunwald and Chang, 1979).
2.2.4. Retention of chemically modified cofactors on
electrodes
Polymer-conjugated cofactors were also often immo-
bilized on electrodes for biosensors. For example, PEG-
NAD+ was entrapped in a photo-polymerized poly(vinyl
alcohol) (PVA) matrix with FDH, salicylate hydroxylase
(SHL) between a dialysis membrane and a gas
permeable PE membrane on an electrode for the
determination of formate. PEG-NAD+ could be effec-
tively regenerated by SHL using sodium salicylate and
oxygen as the substrates. However, the working stability
was limited to 7 days due to the inactivation of the
enzymes (Scheper et al., 1987). In another report,
dextran-NAD+ was entrapped with NADH oxidase and
LDH in a photo cross-linked PVA bearing styrylpyr-
idinium groups on a platinum electrode and was then
covered with a dialysis membrane of cut-off Mw of
10,000. This sensor was reported to function for months
for the detection of d-lactate (Montagné and Marty,
1995). An ethanol biosensor was also developed
following a similar procedure (Leca and Marty, 1997a,
b). A polyurethane hydrogel layer deposited on the
electrode was also investigated for retaining PEG-
NADH and ADH followed by the covering of a dialysis
membrane. The regeneration of PEG-NADH was
catalyzed by a series of the qiunonoid cationic redox
dyes immobilized in graphite-epoxy composite electro-
des (Gründig et al., 1995).
3. Solid-phase cofactors
Membrane entrapment is faced with the technical
dilemma of balancing the retention of catalyst system
and the feeding of substrates. That is probably a
dilemma for any immobilization strategies. Solid-
immobilized catalysts do not need membranes, but
basically has to face the same problem, although the
overall performance of the reactors can be leveraged via
a different set of factors. Nevertheless, solid support-
attached insoluble cofactors are probably easier to reuse
and may afford more flexible reactor design as
compared to dissolved free cofactors, and for that, it
has been explored about as vigorously as membrane
entrapment. Both physical and chemical approaches
have been developed for solid-phase cofactors.
http://www.paper.edu.cn
Fig. 6. Configurations of physical attachment of cofactors to insoluble
solid supports. (a) Surface-adsorbed cofactors; (b) cofactor entrapment
in porous materials; (c) entrapment in cross-linked polymeric
networks.
3.1. Physically immobilized cofactors
3.1.1. Physical attachment of cofactor for organic
synthesis
Cofactors physically immobilized to insoluble solids
have been used by many researchers for organic
synthesis. Similar to enzyme immobilization, cofactors
can be absorbed to the outer surface of solid supports,
entrapped inside porous materials, or contained in cross-
linked polymeric networks (Fig. 6). Surface adsorption
may work best in nonaqueous reaction medium where
the cofactor does not have a good solubility. It was
shown that NAD(H) along with horse liver ADH
(HLADH) coated onto glass beads and were then used
as catalysts for the asymmetric reduction of 2-phenyl-
propionaldehyde and 2-chlorocyclohexanone to their
corresponding ketones in a water-immiscible organic
solvent such as isopropyl ether and ethyl acetate
(Grunwald et al., 1986). The cofactor was regenerated
by the oxidation of ethanol or the reduction of
isobutyaldehyde with TTN of up to 106. Parida et al.
employed partially hydrated porous silica particles (with
a water content corresponding to 70% of pore volume)
for the same enzyme/cofactor system, and it was found
that the system catalyzed the reduction of 2-methyl-
valeraldehyde with a TTN of 3.4  105 and a reaction
rate that was about 6-fold higher than that with
nonporous glass beads (Parida et al., 1992). Wong et
al. entrapped enzymes and cofactor in the matrix of a
cross-linked neutral polyester, XAD-8. The entrapped
enzymes and cofactor was then suspended in an organic
solvent such as butyl acetate containing the substrates
for the synthesis of chemicals. In one example, co-
entrapped HLADH and NADH catalyzed the production
of l-lactaldehyde dimethyl acetal from pyruvaldehyde
dimethylacetal with cyclohexanol as a co-substrate for
cofactor regeneration. A TTN of 80 was achieved in
5 days (Wong, 1986). Recently, NADPH and ADH from
L. kefir (LKADH) were entrapped in PVA gel beads and
were used to transform a number of hydrophobic
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W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384
ketones to their corresponding enantiomerically pure
(R)-alcohols. Cofactor regeneration was achieved within
the beads with isopropanol as the co-substrate catalyzed
by the same enzyme. In the synthesis of (R)-pheny-
lethanol from acetophenone, the TTN of the cofactor
was reported to be up to 103 (De Temiño et al., 2005).
3.1.2. Physical attachment of cofactors on electrodes
Cofactors have been physically immobilized onto
biochemical electrodes through various methods. The
method that applied membranes for retention of free
cofactors has been discussed in Section 2. In those cases,
the cofactors are still “mobile” locally once its sur-
rounding environment is filled with water. Here we
review the methods that apply cofactors physically
attached to a solid-phase support that is insoluble in a
solution.
Surface-attachment is one of the popular methods.
For certain disposable biosensor, enzyme and cofactors
can be physically adsorbed onto the surface of
electrodes (usually modified to enhance their affinity
to enzymes and cofactors) and thus construct simple
biosensors. By a simple drop-coating procedure, GLDH,
2-oxoglutarate and NADH were coated on an MB-
doped screen-printed carbon electrode (SPCE), and the
resulting device could be used as a disposable NH4+
biosensor, which could keep active for 29 days when
stored at 4 °C in a desiccator (Hart et al., 1999).
Similarly, biosensors using NAD+ and corresponding
enzymes for lactate (Sprules et al., 1996) and blood
ketones (Li et al., 2005) were reported. Nakamura et al.
(1996) applied PEI-ferrocene on the surface of graphite
carbon electrode first, then adsorbed glutathione
reductase (GR), G6PDH, and sodium alginate modified
NADP+ (Alg-NADP+) onto the electrode surface.
Matrix-entrapment is another popular physical im-
mobilization method. The typical strategy is to absorb
enzymes and cofactors onto solid powders, and then
form a matrix by physical compression or chemical
reactions. Carbon powder has been used extensively in
the construction of enzyme electrodes. Typically,
enzymes, cofactors, as well as mediators can be mixed
into a carbon paste with the aid of pasting liquids, and
thereby the paste matrix can act as a reservoir of
cofactors. This approach offers good flexibility of
incorporating cofactors, enzymes and other possible
components (Mello and Kubota, 2002). Electron
transfer mechanism in such modified carbon paste
electrodes is not quite clear yet (Habermüller et al.,
2000). Nevertheless, cofactors contained in the paste
were generally still mobile and could diffuse away,
leading to short lifetime of biosensors (Boujtita and El
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377
Murr, 1995). For example, a glucose biosensor based on
carbon paste with GDH, NAD+ and a redox mediator
retained only 10% of its original activity after 1 day
(Gorton et al., 1991). To overcome such a drawback, a
polymeric membrane was usually applied to maintain
the integrity of the paste and prevent loss of cofactors
and mediators. Cation-exchanging poly(ester-sulfonic
acid) (Bremle et al., 1991; Persson et al., 1993) and
charged poly(vinyl pyridine) (Fernández et al., 1998)
films were applied for glucose and ethanol sensors.
Membranes bearing negative charges generally do not
work well for the biosensors detecting negatively
charged substrates such as d-lactic acid under certain
pH condition due to electrostatic expulsion (Bremle et
al., 1991). In addition, membrane coverage of carbon
pastes usually led to significant loss of the sensitivity of
the sensors due to the great mass transfer resistance of
the membranes (Chi and Dong, 1994; Shu et al., 1995).
Various other methods have also been developed to
keep the carbon powder paste integral on electrodes.
Polyelectrolytes, such as PEI, were applied to hold
enzymes and cofactors together within carbon powder
pastes (Domínguez et al., 1993). Cross-linking of
enzymes can stabilize the enzymes significantly, and
help to prevent leakage. It was shown that a carbon paste
electrode consisting NAD+ entrapped within a gel
formed by cross-linking ADH and bovine serum
albumin (BSA) with GA retained 95% of its original
activity after 7 months (Boujtita and El Murr, 1995).
More recently, this method was applied to a lactate
biosensor (Pereira et al., 2006). Formation of powder–
polymer composites is another approach to stabilize the
enzyme/cofactor matrix (Katrlík et al., 1998, 1999).
Reviews on the application of rigid carbon-polymer
composite materials in electrochemical sensing are
available (Alegret, 1996; Cespedes and Alegret, 2000).
3.2. Chemical incorporation of cofactors
Tethering cofactors to solid supports can effectively
prevent leakage of cofactors. However, chemical
modifications may lead to poor cofactor activity as it
may alter the binding affinities of cofactors to the active
sites of enzymes. In one of the earlier works, it was
demonstrated that NAD+ immobilized on diazotized
glass beads exhibited redox activity with YADH for the
production of acetaldehyde from ethanol. Regeneration
of NAD+ was achieved by using acetaldehyde with the
same enzyme, however, TTN of NAD+ was only around
1 (Weibel et al., 1971). NAD+-N6-[N-(6-aminohexyl)-
acetamide] was attached to Sepharose 4B through
cyanogen bromide group and showed an activity with
中国科技论文在线 http://www.paper.edu.cn
378 W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384

Fig. 7. Chemical attachment of enzyme and cofactor on the surface of nylon tube.

free YADH or LDH that was less than 1% of the free
cofactor (Larsson and Mosbach, 1971). Regeneration of
such immobilized cofactor could be achieved with LDH
using pyruvate as the substrate (Lindberg et al., 1973).
While most of the solid-attached cofactors were
active with free enzymes, it has been difficult to achieve
activity with systems that have cofactor and enzymes
both immobilized. Wykes et al. reported that Agarose-
attached NAD+ was active with free ADH and LDH,
whereas no activity was observed with ADH and LDH
immobilized separately on DEAE-cellulose matrices
(Wykes et al., 1975). NADH and HLADH co-
immobilized on Sepharose 4B showed certain activity,
but the immobilized cofactor could not be regenerated
either enzymatically with a pyruvate/LDH system or
chemically due to steric hindrance (Gestrelius et al.,
1975). Mazid et al. co-immobilized NAD+ and YADH
on the surface of nylon tube (Fig. 7) with the cofactor
was regenerated chemically in the presence of an
indophenol dye (Mazid and Laidler, 1982). It was
claimed that the enzyme retained about 10% of its
activity.
Success in regeneration of co-immobilized cofactors
was achieved by using a coupled-substrate regeneration
strategy with single-enzyme systems. HLADH and
NAD(H) was covalently incorporated into cross-linked
albumin porous particles by GA cross-linking and used
for the oxidation of alcohols, the cofactor was
regenerated by supplying different substrates (Pulvin
et al., 1986; Lortie et al., 1989). Similarly, cross-linked
HLADH–NADH crystalline complex was used in the
production of cinnamyl alcohol from cinnamaldehyde,
and ethanol was supplied as a co-substrate for cofactor
regeneration (Lee et al., 1986). Recently, the same
enzyme and cofactor complex was applied to the
reduction of 6-methyl-5-hepten-2-one and several
ketones with isopropanol replacing ethanol as the
cofactor regenerating substrate (Clair et al., 2000). The
single enzyme–cofactor strategy was applied for
development of biosensors where the cofactor can be
regenerated electrochemically. An NAD+-analog was
co-immobilized with LDH on the surface of a porous
glass carbon electrode to give an amperometric lactate
biosensor (Khayyami et al., 1996). NAD+ was regen-
erated by direct electrochemical oxidation or with a
soluble mediator, MB. In another work, N6-(2-ami-
noethyl)-NAD+ was covalently attached to the pyrrolo-
quinoline quinone (PQQ)-monolayer-functionalized
electrode, and then NAD+-dependent LDH or ADH
was associated to the PQQ-NAD+ monolayer through

Fig. 8. Chemical attachment procedures of enzyme and cofactor on pyrroloquinoline quinone-functionalized electrode.
中国科技论文在线 http://www.paper.edu.cn
W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384 379

Fig. 9. Chemical incorporation of enzymes and cofactor into nano-porous silica glass. Although being tethered to the wall of silica glass pores,
enzymes and co-enzyme can still share interactions via thermal vibration due to the close vicinity within the nanopores.

affinity interactions. Upon cross-linking, an integrated
electrically-wired enzyme electrode was constructed
(Fig. 8). The cross-linked enzyme layer exhibited high
stability for alcohol detection (Bardea et al., 1997). In
the development of glucose sensors, cofactor FAD was
first “wired” to electrode via conductive tethers
followed by affinity binding of apo-glucose oxidase
(Katz et al., 1997; Willner et al., 1996; Xiao et al., 2003).
A similar immobilization and regeneration strategy of
cofactor was applied in an integrated NAD+-dependent
enzyme field-effect transistor device for the biosensing
of lactate (Zayats et al., 2000).
Much less work has been reported for the regener-
ation of immobilized cofactors with co-immobilized
multi-enzyme systems. Yamazaki and Maeda reported a
work with covalently immobilized NAD but with
physically entrapped enzymes (Yamazaki and Maeda,
1982). Pre-modified NAD+ was co-polymerized into a
poly(acrylamide) hydrogel matrix in the presence of
native enzymes. In this way, co-immobilized multi-
enzyme systems, FDH–MDH–NAD+ or HLADH–DI–
NAD+, were constructed. Regeneration of the cofactor
was achieved, and its activity could keep for several
days. Ukeda et al.'s applied GA to co-cross-link NAD+,
YADH and DI onto Sepharose, and a mean cycling rate
of 8 h− 1 was obtained for the cofactor (Ukeda et al.,
1989a,b). The use of nanostructures provides a new
route to realize co-immobilized multi-enzyme/cofactor
bioactive systems. Recently El-Zahab et al. applied
nano-porous silica glass as the support of both enzymes
and cofactor NADH (Fig. 9) (El-Zahab et al., 2004).
LDH and a regenerating enzyme GDH were co-

Fig. 10. Nanoparticle-driven multi-enzyme catalytic systems in form of an artificial cell. Nanoparticles carrying surface-attached enzymes and
cofactor will provide enzyme–cofactor interactions within the porous capsule (∼100 μm in diameter) through particle collision.
中国科技论文在线 http://www.paper.edu.cn
380 W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384

Table 1 4. Conclusion
Characteristics of cofactor regeneration strategies
Methods of cofactor Concerns Advantages Biocatalysis can offer highly selective reactions,
retention environmentally friendly processes, and energy-effec-
Membrane entrapment tive operations, as compared to traditional chemical
Free cofactor Cofactor leaching; Easy to practice; processing. The widespread public concerns about
via size exclusion limited reactor options; maintain the
high mass transfer intrinsic activity
environmental quality and energy resources have made
resistance of the bioprocessing increasingly desirable to many industrial
catalyst system applications such as chemical production, drug synthe-
Free cofactor pH and ionic strength Reduce mass sis, environmental remediation, and fuel refining.
via electrostatic of solution may impact transfer rates Biosynthesis may be achieved with either whole cells
interaction overall retention of substrates
effectiveness; while maintain
or isolated enzymes. Often the cellular processes are
limited mass transfer the intrinsic associated with slow reactions and complicated culturing
of polar chemicals activity of conditions. Significant advances have been made
the catalyst through genetic and/or metabolic engineering for
Chemically Activity loss of Allow the use industrial microbial technology; but that approach raises
modified cofactor modified cofactor of membranes
of larger pores
concerns over the release of non-natural organisms into
for reduced the environment. On the other hand, enzymatic bioca-
mass transfer talysis is faster, cleaner and simpler. The current arts of
resistance enzymatic biosynthesis are primarily for single-enzyme
Solid attachment systems. How to apply multi-enzyme systems to carry
Physical Cofactor leaching; Flexible reactor
adsorption or mass transfer design; easy to
out complex reactions, especially those involve cofac-
entrapment resistance of substrates practice; easy tors, represents one great challenge in bioprocessing.
replacement of Apparently this will also be a long-standing challenge.
lost catalyst Although decades of research and development efforts
Chemical Activity loss of Flexible reactor have been made, no ideal solutions have emerged. All the
tethering modified cofactor ; design; may
difficult to achieve stabilize the
strategies reviewed in this paper have mixed advantages
shuttling between catalyst system; and disadvantages. Table 1 summarizes a simple com-
two co-immobilized reduced mass parison of the characteristics of different methods for
enzymes transfer limitations cofactor retention. Generally speaking, membrane en-
trapment has the advantage to allow the use of free
immobilized along with the cofactor via spacers of cofactors that have excellent mobility and high activity,
different sizes. The coupled reactions catalyzed by LDH but is compromised by small membrane pore size as
and GDH were achieved, indicating the immobilized determined by the size of the cofactor. A similar problem
NAD(H) shuttled between the two co-immobilized is faced with solid-phase cofactors when the cofactors are
enzymes. It was also found that reaction rate increased physically entrapped or adsorbed. Such configurations
when longer spacer and smaller pores were employed. It will lead to mass transfer-limited reaction kinetics, and
was believed that the pores helped to bring together all should be considered for reaction systems that have low
the catalytic components, while thermal vibration of intrinsic enzyme activities. Immobilization of cofactors to
molecules facilitated the shuttling of the cofactor solid supports through covalent bonds may exhibit
between the two enzymes. In a different approach, advantages of materials with much more open structures
enzymes and cofactor chemically attached onto nano- to minimize mass transfer resistance, but the chemical
particles were encapsulated into polymeric capsules modification may significantly reduce the activity of the
(Wang et al., 2005). The membrane of the capsules was catalyst system. Such a strategy can lead to reaction-
porous with pore size controlled to be in nanometer limited kinetics, and should be considered for catalyst
scale to allow substrates and products to penetrate while systems that have high intrinsic activities. The micro-
retain the particle-attached enzymes and cofactor. Since capsulation of nanoparticle-attached cofactor and
the nanoparticles were mobile inside the capsules when enzymes was designed in an attempt to combine the
they were filled with water, collision between particles advantages of membrane entrapment and solid-attach-
could enable interactions among the enzymes and ment (Wang et al., 2005). It can be expected that new
cofactor, thus allowing multiple reactions to take place approaches and concepts will continue to emerge, and
inside the capsules (Fig. 10). most probably will through the introduction of more
中国科技论文在线
W. Liu, P. Wang / Biotechnology Advances 25 (2007) 369–384
sophisticated structures and materials for improved
activity and retention of the cofactors.
Acknowledgements
The authors thank the National Natural Science
Foundation of China (NSFC#20576135) and the Na-
tional Knowledge Innovation Project from the Chinese
Academy of Sciences (# KSCX2-YW-G-019) and 863
Project (#2006AA02Z217) for the financial support.
P.W. acknowledges the support of a CAREER award from
the US National Science Foundation (BES# 0348412).
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