Journal of Biotechnology 139 (2009) 102–107
Contents lists available at ScienceDirect
Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec
Nanoparticle-supported multi-enzyme biocatalysis with in situ cofactor
regeneration
Wenfang Liu a,b , Songping Zhang a , Ping Wang c,∗
a
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, China
School of Chemical Engineering and Environment, Beijing Institute of Technology, Beijing 100081, China
c
Department of Bioproducts and Biosystems Engineering and Biotechnology Institute, University of Minnesota, 2004 Folwell Ave., 208 Kaufert Lab, St. Paul, MN 55108, United States
b
a r t i c l e
i n f o
Article history:
Received 17 March 2008
Received in revised form
29 September 2008
Accepted 29 September 2008
Keywords:
Nanoparticles
Enzyme immobilization
Multi-enzyme biotransformation
Cofactor regeneration
Bioprocessing
Biosynthesis
a b s t r a c t
Although there have been a long history of studying and using immobilized enzymes, little has been
reported regarding the nature of immobilized cofactors. Herein we report that cofactor NAD(H) covalently attached to silica nanoparticles successfully coordinated with particle-immobilized enzymes and
enabled multistep biotransformations. Specifically, silica nanoparticle-attached glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH) and NAD(H) were prepared and applied to catalyze the coupled
reactions for production of ␣-ketoglutarate and lactate with the cofactor regenerated within the reaction
cycle. It appeared that particle–particle collision driven by Brownian motion of the nanoparticles provided effective interactions among the catalytic components, and thus realized a dynamic shuttling of
the particle-supported cofactor between the two enzymes to keep the reaction cycles continuing. Total
turnover numbers (TTNs) as high as 20,000 h−1 were observed for the cofactor. It appeared to us that the
use of particle-attached cofactor promises a new biochemical processing strategy for cofactor-dependent
biotransformations.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
Cofactor-dependent oxidoreductases constitute an important
group of enzymes that promise highly selective synthesis of chiral
compounds such as chiral alcohol, hydroxyl acids or amino acids
(Hummel, 1999). Naturally occurring cofactors like NAD(H) and
NADP(H) generally act as electron carriers facilitating such redox
biotransformations and are consumed at stoichiometric ratios.
Native cofactors are often much more expensive than the enzymes
and their regeneration and reuse are critical for any preparative applications. Compare to other regeneration methodologies
such as chemical and electrochemical reactions, enzyme-catalyzed
cofactor regeneration offers selective and mild reactions and has
been pursued extensively. Such enzymatic regeneration generally
requires a cofactor effectively shuttle among different enzymes
to keep the regeneration cycles continuing. Although enzymatic
regeneration of free cofactors have been demonstrated for many
biotransformation reactions, it has been a long-standing challenge
in realizing cofactor regeneration with the enzymes and cofactor
both immobilized (Liu and Wang, 2007).
∗ Corresponding author. Tel.: +1 612 624 4792; fax: +1 612 625 6286.
E-mail address: [email protected] (P. Wang).
0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2008.09.015
Nevertheless, several different approaches have been explored
to realize cofactor regeneration in immobilized reaction systems.
Cofactor retention by using microcapsules (Chang et al., 1982; Ilan
and Chang, 1986; Yu and Chang, 1981a,b, 1982) or membrane reactors (Howaldt et al., 1990; Ikemi et al., 1990a,b; Kitpreechavanich
et al., 1985; Kulbe et al., 1990; Lin et al., 1997, 1999; Nidetzky et
al., 1996, 1994; Obón et al., 1996, 1998; Röthig et al., 1990) has
been the most widely adopted method. The cofactors essentially
act in form of free cofactors in such membrane-restricted reaction systems. However, it has been always challenging to balance
between the permeability of the substrates or products and the
retention of the enzymes and cofactors, especially with cofactors
of small sizes. Enlarging the size of cofactors by chemical modification with water soluble polymers can alleviate this problem to
certain extent (Davies and Mosbach, 1974; Devaux-Basseguy et al.,
1997; Furukawa et al., 1980; Larsson and Mosbach, 1974; Stengelin
and Patel, 2000; Wandrey and Bossow, 1986; Yamazaki et al., 1976);
however, such modifications may also deactivate cofactors (Larsson
and Mosbach, 1974; Zappelli et al., 1976, 1975).
Compare to membrane-contained systems, solid supportattached insoluble cofactor and enzymes are much easier to
reuse and may afford more flexible reactor design. Early trials with support-attached cofactor–enzyme systems generally
failed. Wykes et al. (1975) prepared immobilized NAD+ by coupling it with a hexamethylamine derivative of agarose using
W. Liu et al. / Journal of Biotechnology 139 (2009) 102–107
dicyclohexyl-carbodiimide, while lactate dehydrogenase (LDH) and
yeast alcohol dehydrogenase (YADH) were immobilized on DEAEcellulose matrices. The results showed that both immobilized
enzymes and immobilized cofactor were active when they were
tested separately with their free counterparts, but no activity
could be detected with immobilized NAD+ and immobilized dehydrogenases. In another effort, Gestrelius et al. co-immobilized
NADH along with horse liver alcohol dehydrogenase (HLADH) on
cyanogen bromide-activated Sepharose 4B to catalyze the reduction of lactaldehyde (Gestrelius et al., 1975). The immobilized
system successfully catalyzed the desired reaction and the cofactor
was also able to be regenerated with the same enzyme by feeding
the reaction system with a secondary substrate, ethanol. However,
the cofactor could not be regenerated when a second enzyme LDH
was applied. In a later work, LDH was co-immobilized by using glutaraldehyde (GA) to Sepharose-bound NAD+ –HLADH complex, and
moderate cofactor regeneration activity was reported with such an
immobilized system (Månsson et al., 1979).
A successful example for immobilized enzyme-coupled regeneration of immobilized cofactor was reported by Yamazaki and Maeda
(1982). An NAD+ derivative carrying a vinyl group was copolymerized into a polyacrylamide gel inside which enzymes were
physically entrapped. The rate of cofactor regeneration of such an
immobilized system was reported to be 14 h−1 . Recently, cofactor
and enzymes were covalently immobilized utilizing nano-porous
structure of porous silica glass (El-Zahab et al., 2004). LDH and
a regenerating enzyme glucose dehydrogenase (GDH) were coimmobilized along with NADH via spacers of different sizes. The
coupled reactions catalyzed by the co-immobilized LDH and GDH
were achieved, indicating immobilized NAD(H) shuttled between
co-immobilized enzymes. However, recycling rate of cofactor was
still low (with total turnover number (TTN) up to 102).
All the above approaches have been developed by confining the
catalyst systems into a limited space. Again, that is likely to be associated with mass transfer limitations. One alternative approach is to
use particle-immobilized systems, where active enzyme–cofactor
interactions can be realized through particle–particle collisions,
while the reaction system is more open to substrates in the reaction media (Wang, 2006). Mobile nanoparticle systems based on
hetero-bifunctional polymer particles (Du et al., 2005) and artificial
nanotransport systems created from microspheres and motor protein (Yokokawa et al., 2008) have been successfully demonstrated.
The mobility of nanoparticles were also applied to enable multistep biotransformations in a recent work with polystyrene particle
(500 nm in diameter)-attached cofactor NAD(H) along with four
enzymes that were also immobilized on particles for the synthesis of methanol from carbon dioxide (El-Zahab and Wang, 2008).
However, only moderate TTN was achieved (<1 h−1 ). In a previous
study we have demonstrated that the size of particles can impact
the activity of the surface-attached enzymes by controlling the frequency of collision (Jia et al., 2003), and smaller particles could
afford higher specific enzyme activities because of their higher
mobility. In the current work, we apply silica nanoparticles of 30 nm
in diameter as the supports to immobilize enzymes and cofactor to
examine the effectiveness of cofactor regeneration with particleattached systems. As a model system, glutamate dehydrogenase
(GLDH) and lactate dehydrogenase and their cofactor NAD+ were
immobilized separately on the nanoparticles for this study.
103
1.1.1.27, Type XI, salt-free lyophilized powder), NAD+ and NADH
were purchased from Sigma Chemical Co. (St. Louis, USA). Sodium
pyruvate and l-glutamic acid were purchased from Acros Organics
(Gell, Belgium). The latter was neutralized with sodium hydroxide before using. Silica nanoparticles with an average diameter of
30 ± 5 nm and a specific surface area of 200 ± 20 m2 /g were purchased from Zhjiang Hongsheng Science & Technology (Zhoushan,
China). ␥-Glycidoxypropyl trimethoxysilane (GPS) was provided as
a gift from Nanjing Shuguang Chemicals (Nanjing, China). Other
chemicals including toluene, triethylamine (Et3 N), acetone, sodium
phosphate, citric acid, triethanolamine, hydrochloric acid, sodium
hydroxide, NH4 Cl, NH4 OH, Tris and HEPES were all of analytical
grade.
2.2. Activation of silica nanoparticles
Silica nanoparticles were activated according to modified procedure as reported previously (Lin et al., 2001). A typical procedure
included the addition of 1 g silica nanoparticles into 49 mL toluene.
After sonication for 30 min, 0.15 mL Et3 N and 1 mL GPS were then
added into the suspension. The activation reaction was carried out
with reflux and stirring at 115 ◦ C for 3 h under N2 atmosphere. After
being air-cooled to room temperature, the particles were recovered
via filtration and then washed with acetone for several times. The
amount of epoxide groups tethered to the particles was determined
by an acid–base titration method (Sundberg and Porath, 1974). The
titration was typically conducted with a hydrochloric acid after
reacting the activated and dried silica nanoparticles (0.5 g) with
5 mL sodium thiosulfate solution (1.3 M) for 2 h. The loading of
epoxide groups was measured to be 0.6 mmol/g-particle.
2.3. Immobilization of enzyme and cofactor
2. Materials and methods
Enzymes were immobilized onto the silica particles by forming
covalent bonds through the epoxide group on the surface of the
particles. Typically 100 mg silica and 2.4 mg enzyme (either GLDH
or LDH) were mixed in 1.5 mL pH 7.5 phosphate buffer (0.1 M) at
4 ◦ C under shaking (200 rpm). The reaction was allowed to last 24 h
before the particles were removed from the solution via centrifugation (13,000 × g). The particles were washed at least three times
with 25 mL of 0.1 M Tris–HCl buffer (pH 7.5) solution each time till
no protein was detected in the washing solution. Enzyme loading
on the particles was determined based on the difference of the protein content in the solution (including the washing solution) before
and after immobilization as measured using Bradford assay. Typical loading of GLDH was found to be 13 ± 0.2 mg-enzyme/g-particle,
while that for LDH was 20 ± 0.2 mg/g. These results are comparable to those reported previously for enzymes covalent attached to
nanostructures (El-Zahab et al., 2004; Kim et al., 2006), yet lower
than those physically absorbed onto polymeric beads where loadings around 4.4% (w/w) were achieved (Le and Means, 1998).
The procedures for cofactor (NAD+ ) immobilization and subsequent purification were similar to that of the enzymes except the
materials were applied with a different ratio: 10 mg cofactor was
added for each 5 mg activated silica particles. The modified particles were also washed using 1 M NaCl solution in addition to the
washing with buffer solution in order to remove any physically
absorbed cofactor molecules. Immobilized loading of NAD+ was
found to be rather high, 0.4 g-cofactor/g-particle, approaching 100%
of the epoxide group on the surface of the particles.
2.1. Materials
2.4. Enzymatic reactions
GLDH from bovine liver (EC: 1.4.1.3, Type III, lyophilized powder with a protein content of 70%), LDH from rabbit muscle (EC:
Unless specified otherwise, enzymatic reactions were conducted in 0.1 M Tris–HCl buffer (pH 7.5) at room temperature under
104
W. Liu et al. / Journal of Biotechnology 139 (2009) 102–107
Fig. 1. A postulated reaction mechanism for silica nanoparticles-supported enzyme–cofactor catalyst system (NAD+ and NADH: oxidized and reduced format of the cofactor
nicotinamide adenine dinucleotide; GLDH: glutamate dehydrogenase; LDH: lactate dehydrogenase).
shaking (130 rpm), with the amount of each catalytic component
was controlled to reach the desired molar ratio. A typical reaction
may include the use of 10 mg GLDH-carrying particles, 1 mg LDH
nanoparticles and 0.4 ␮g NAD+ -particles applied in 0.8 mL reaction solution containing 20 mM glutamate and 10 mM pyruvate.
The reaction rate of the couple reactions was monitored by measuring the production rates of the reaction products. Aliquots of 80 ␮L
were taken periodically for HPLC analysis after centrifugation at
20,000 × g for 10 min to remove the catalysts. A Waters HPLC system equipped with a Supelcosil C-18 column (250 mm × 4.6 mm)
was applied with 0.05 M sodium phosphate solution of pH 3.0 as the
mobile phase at a flow rate of 0.5 mL/min. Analyses were conducted
with triplicates, standard errors were generally within 5%. As the
characterization of particle’s dispersion in the reaction solution, the
effective diameter of silica nanoparticles suspended in the solution
was measured using a dynamic laser light-scattering particle size
analyzer (Brookhaven 90Plus, USA).
3. Results and discussion
3.1. Coupled reactions with in situ regeneration of
particle-attached NAD(H)
Fig. 1 depicts the postulated interactions among the enzymes
and cofactor involved in the reaction system. Cofactor regeneration cycle is realized through the action of two enzymes, LDH and
GLDH. The translocation of the cofactor between the two enzymes
is essential to the desired coupled reactions. Free enzymes and
cofactors generally assume good Brownian motion mobilities in
a homogeneous reaction environment for efficient biotransformations. Nanoparticle-attached enzymes, albeit being immobilized on
insoluble solid supports, may possess similar mobilities through
the Browniang motion of the particles and thus enable reactions
with velocities that are in some cases close to those of homogeneous reactions with free enzymes (Jia et al., 2003). For the current
reaction system, the mobility of particles is expected to generate particle–particle interactions that allow the surface-exposed
cofactor coordinate with the enzymes and thus driving forward
the reactions. The silica nanoparticle-supported enzyme–cofactor
system examined in this work appears to follow such a reaction
mechanism. Data reported in Fig. 2 compare the reaction system to
soluble free enzymes and cofactor. To avoid possible leaching of the
enzymes and cofactor from the particles, an extensive washing procedure including the use of salted solutions has been applied during
the preparation of the immobilized catalysts. It was also observed
through our analytical procedure that reactions were effectively
stopped upon the removal of silica particles via centrifugation,
further supporting the observed activities of the particle-attached
enzymes and cofactor.
The reaction rates achieved with the immobilized catalyst system were comparable to those of free catalyst system when the
initial concentration of the cofactor was low (Fig. 2). However,
the maximum reaction velocity (vmax ) of the immobilized system
was only about 50% of that of the free system. Unlike the free
catalyst system, the immobilized catalyst reach cofactor saturation at a very low cofactor concentration (1 ␮M), indicating a low
apparent Michaelis constant (KM ). Interestingly, the immobilized
system showed a higher vmax than the reactions with either only the
enzymes or the cofactor was immobilized. Since the particles were
also able to adsorb free enzymes or cofactor, we believe the slowed
reactions of the partially immobilized catalyst systems have been
resulted from the reduced availability of the free catalytic components in the solution. The silica particles can generally absorb a
good amount of enzymes or cofactor onto their surfaces. Although
certain binding sites have been occupied with covalently attached
catalytic species, there are still binding sites open on the particles
for physical adsorption. Those physical binding sites were able to
absorb enzymes and cofactor during the immobilization procedure;
however, subsequent washing and purification steps after immobilization will then leave those sites open again for future adsorption.
Fig. 2. Effect of initial cofactor concentration on reaction velocity. Volume of reaction solution was controlled to be 0.8 mL, the amount GLDH- and LDH-attached
particles were 0.13 and 0.02 mg; and the concentrations of glutamate and pyruvate
were 20 and 10 mM, respectively. Reaction velocity is defined as ␮mol-product/min.
W. Liu et al. / Journal of Biotechnology 139 (2009) 102–107
105
Table 3
Effect of buffer solution on the TTN of particle-attached NAD(H). Reactions were
conducted with particles carrying an equivalent amount of 0.3 ␮M of cofactor. Both
substrates, glutamate and pyruvate, were applied at a concentration of 30 mM. Reactions were allowed to last 2 h. For all buffer solutions examined were set for pH 7.5
with a total salt concentrations as 0.1 M.
Buffer systems
TTN
Na2 HPO4 –NaH2 PO4
Na2 HPO4 –KH2 PO4
Na2 HPO4 –citric acid
Triethanolamine–HCl
Tris–HCl
NH4 Cl–NH4 OH
HEPES–NaOH
Fig. 3. Total turnover number (TTN) of particle-attached cofactor NAD(H) as a function its initial concentration. (TTN was determined for a reaction time of 8 h, defined
as moles of product normalized with respect to the moles of cofactor applied. Reaction conditions are the same as that in Fig. 2.)
Examination on the total turnover number of the cofactor
achieved with the particle-immobilized catalyst system indicated
that very dynamic cofactor–enzyme interactions have been realized. As shown in Fig. 3, TTN for a reaction time of 8 h reached
as high as 2800 (or equivalent to ∼350 h−1 ) when the initial concentration of the cofactor was low (0.3 ␮M). Since the reaction
reaches cofactor saturation at a low NAD+ concentrations, increasing the amount of NAD+ did not improve the overall reaction rate,
but led to TTNs decreasing with a ratio proportional to the reciprocal of the cofactor concentration (Fig. 3). However, higher TTNs
can be achieved by increasing the concentrations of the substrates
or the amount of enzymes. Table 1 summarizes the data of TTN
for a reaction time of 1 h but with much higher concentrations of
enzymes and substrates. When glutamate and pyruvate concentration both increased to 30 mM, a TTN of 15,700 h−1 was achieved.
Further increase in substrate concentration led to decreased percentage conversions of the substrates, indicating the reaction was
approaching substrate saturation.
1,200
740
450
16,000
18,000
6,700
17,000
We further examined the effect of enzyme ratio on the efficiency of cofactor regeneration by fixing the concentration of
silica-attached NAD+ at 0.3 ␮M. As shown in Table 2, both initial
reaction rate and the TTN of NAD+ decreased as the GLDH increased.
This result was surprising since additional amounts of enzymes
should not inhibit the reaction even if not promote the reaction.
Apparently other factors instead of enzyme ratio are predominant
at the current reaction conditions. As to be discussed in the following, the total amount of particles in the solution has in fact
a stronger impact on the performance of the particle-supported
reaction system.
3.2. Factors that control the performance of particle-attached
NADH
The physicochemical properties of the reaction solution may
have a strong impact on the particle-supported catalyst system.
For that, we especially examined the effect of the properties of
buffer solution and total amount of particles, as both may impact
the dispersion, aggregation and motion of the particles. Table 3
shows the effect of different types of buffer systems set at pH 7.5 on
the TTN of silica-attached NAD(H). As a result, TTN was generally
low when phosphate buffer solutions were applied, similar to the
observations reported by others for NAD(H) with phosphate buffer
solutions (Chenault and Whitesides, 1987). However, high TTNs
(above 15,000 within 2 h) were observed with other buffer systems
tested with Tris–HCl buffer gave the highest TTN. We further examined the effect of pH value and the concentration of the Tris–HCl
buffer system. Within the range of pH values tested (varied from
Table 1
TTN of the coupled reactions with particle-attached cofactor and enzymes with different substrate concentrations. The reactions were conducted with 10 mg GLDH particles,
1 mg LDH particles, NAD particles with amount of cofactor equivalent to 0.3 ␮M in 0.8 mL buffered reaction solution for 1 h.
Pyruvate (mM)
Glutamate (mM)
Initial reaction rate (␮mol/min)
2.5
5
10
15
20
25
30
30
20
20
20
20
20
20
20
30
0.019
0.030
0.070
0.120
0.227
0.433
0.460
0.418
TTN of silica-supported NAD+
585
1,100
3,290
6,600
9,500
14,200
15,000
15,700
Glutamate conversion (%)
Pyruvate conversion (%)
1.0
1.8
5.5
11.0
15.9
23.7
25.1
17.4
7.8
7.3
11.0
14.7
15.9
19.0
16.7
17.4
Table 2
Effect of mass ratio of particle-attached enzymes on the TTN of the cofactor. The reactions were conducted with NAD particles of an amount equivalent to 0.3 ␮M cofactor,
20 mM glutamate and 10 mM pyruvate in 0.8 mL buffered reaction solution for 1 h.
Silica bearing GLDH (mg)
Silica bearing LDH (mg)
Mass ratio of GLDH to LDH
Initial reaction rate (␮mol/min)
TTN of silica-attached NAD+
1
5
10
20
1
1
1
1
0.6
3.2
6.5
13.5
0.097
0.079
0.070
0.058
4200
3600
3300
2300
106
W. Liu et al. / Journal of Biotechnology 139 (2009) 102–107
Fig. 4. Effect of pH value on the TTN of particle-attached NAD(H) with particleattached enzymes. The reactions were conducted with 0.1 M Tris–HCl buffer solution
for 1 h. Volume of reaction solution was 0.8 mL with GLDH- and LDH-particles were
applied at 1 mg each; the amount NAD-particles was controlled to maintain a cofactor of 0.3 ␮M; both glutamate and pyruvate were applied at 30 mM.
7.3 to 8.0, determined by the preferred pH of GDH and LDH), the
reaction appeared to be insensitive to changes in pH value (Fig. 4).
Instead, the concentration of Tris salt in the buffer solution showed
a stronger impact (Fig. 5). It appeared lower salt concentrations are
preferred with TTN reached 20,000 h−1 at 10 mM.
Particle mobility is a key factor in realizing the regeneration of
the cofactor and coupled reactions with the immobilized catalytic
system. Higher concentration of particles is generally desired to
improve the collision frequency and thus the overall reaction rate;
however, particles also tend to aggregate at higher concentrations.
Although the surface-attached enzyme and cofactor molecules
improve the compatibility of the particles with the aqueous solution and help them disperse, they are also “sticky” to each other
and aggregation will be more likely to form at higher concentrations. We examined this issue by studying the reaction rate as a
function of concentration of total particles. As shown in Fig. 6, TTN
of the cofactor maintained high when the solid concentration was
low (with liquid–solid mass ratio above 200 in Fig. 6), whereas
it dropped sharply as the solid concentration further increased
Fig. 6. Effect of liquid–solid mass ratio on the TTN of particle-attached NAD(H). ()
TTN of the cofactor realized with 10 mg GLDH particles and 1 mg LDH particles in
various amount of liquid solution (0.5–3 mL, pH 7.5), with all the other reaction
conditions the same as those in Fig. 4. () The effective diameter of particles and
their aggregates in the reaction solution determined via light scattering.
(in the low value region of the liquid–solid mass ratio). That suggested a critical phase transition of the particles took place when
the liquid–solid ratio reduced to 200. Our particle size detection
using light scattering also supported such a transition as the apparent particle size increased sharply as the liquid–solid mass ratio
decreased, indicating severe particle aggregation took place at that
solid concentration region (Fig. 6).
4. Conclusions
GLDH, LDH and their cofactor NAD(H) were covalently immobilized separately on the surface of silica nanoparticles and were
applied successfully for coupled biotransformation reactions with
in situ cofactor regeneration. As expected, the mobility of the particles is critical to realizing dynamic enzyme–cofactor interactions,
and efficient biotransformations can be achieved when the particles well dispersed in the solution. The use of particle-attached
systems will allow easy recovery through methods such as filtration
and precipitation, and can be recycled and reused, thus substantially improving the potential of cofactor-dependent biochemical
reactions for large-scale industrial bioprocessing applications.
Acknowledgements
Liu thanks financial support of a postdoctoral fellowship from
National Science Foundation of China (NSFC#20060390522). The
authors thank supports from NSFC and Chinese Academy of Sciences (NSFC#20576135, #KSCX2-YW-G-019, and #2006AA02Z217
of 863 initiative). Wang acknowledges the support from US National
Science Foundation (BES#0348412). The authors thank Ms. Muqing
Zheng for assistance in some of the experimental work.
References
Fig. 5. Effect of salt concentration on the TTN the particle-attached NAD(H). pH
value was fixed at 7.5 with all the other conditions the same as those in Fig. 4.
Chang, T.M.S., Yu, Y.T., Grunwald, J., 1982. Artificial cells immobilized multienzyme
systems and cofactors. Enzyme Eng. 6, 451–561.
Chenault, H.K., Whitesides, G.M., 1987. Regeneration of nicotinamide cofactors for
use in enzymatic synthesis. Appl. Biochem. Biotechnol. 14 (2), 147–197.
Davies, P., Mosbach, K., 1974. The application of immobilized NAD+ in an enzyme
electrode and in model enzyme reactors. Biochim. Biophys. Acta 370 (2),
329–338.
Devaux-Basseguy, R., Bergel, A., Comtat, M., 1997. Potential applications of NAD(P)dependent oxidoreductases in synthesis: a survey. Enzyme Microb. Technol. 20,
248–258.
W. Liu et al. / Journal of Biotechnology 139 (2009) 102–107
Du, Y.-Z., Hiratsuka, Y., Taira, S., Eguchi, M., Uyeda, T.Q.P., Yumoto, N., Kodaka, M.,
2005. Motor protein nano-biomachine powered by self-supplying ATP. Chem.
Commun., 2080–2082.
El-Zahab, B., Jia, H., Wang, P., 2004. Enabling multienzyme biocatalysis using
nanoporous materials. Biotechnol. Bioeng. 87 (2), 178–183.
El-Zahab, B., Wang, P., 2008. Particle-attached NADH for methanol production from
CO2 catalyzed by coimmobilized enzymes. Biotechnol. Bioeng. 99 (3), 508–514.
Furukawa, S., Katayama, N., Izuka, T., Urabe, I., Okada, H., 1980. Preparation of
polyethylene glycol-bound NAD and its application in a model enzyme reactor.
FEBS Lett. 121 (2), 239–242.
Gestrelius, S., Mansson, M., Mosbach, K., 1975. Preparation of an
alcoholdehydrogenase-NAD(H)-sepharose complex showing no requirement
of soluble coenzyme for its activity. Eur. J. Biochem. 57, 529–535.
Howaldt, M., Kulbe, K.D., Chmiel, H., 1990. A continuous enzyme membrane reactor retaining the native nicotinamide cofactor NAD(H). Ann. NY Acad. Sci. 589,
253–260.
Hummel, W., 1999. Large-scale applications of NAD(P)-dependent oxidoreductases:
recent developments. Trends Biotechnol. 17 (12), 487–492.
Ikemi, M., Ishimatsu, Y., Kise, S., 1990a. Sorbitol production in charged membrane
bioreactor with coenzyme regeneration system. II. Theoretical analysis of a continuous reaction with retained and regenerated NADPH. Biotechnol. Bioeng. 36
(2), 155–165.
Ikemi, M., Koizumi, N., Ishimatsu, Y., 1990b. Sorbitol production in charged
membrane bioreactor with coenzyme regeneration system. I. Selective retainment of NADP(H) in a continuous reaction. Biotechnol. Bioeng. 36 (2), 149–
154.
Ilan, E., Chang, T.M.S., 1986. Lipid-polyamide–polyethyleneimine microcapsules
for immobilization of free cofactors and multienzyme system. Appl. Biochem.
Biotechnol. 13 (3), 221–230.
Jia, H., Zhu, G., Wang, P., 2003. Catalytic behaviors associated with enzymes attached
to nanoparticles: the effect of particle mobility. Biotechnol. Bioeng. 84, 406–414.
Kim, M.I., Ham, H.O., Oh, S.-D., Park, H.G., Chang, H.N., Choi, S.-H., 2006. Immobilization of Mucor javanicus lipase on effectively functionalized silica nanoparticles.
J. Mol. Catal. B: Enzyme 39, 62–68.
Kitpreechavanich, V., Nishio, N., Hayashi, M., Nagai, S., 1985. Regeneration and
retention of NADP(H) for xylitol production in an ionized membrane reactor.
Biotechnol. Lett. 7 (9), 657–662.
Kulbe, K.D., Howaldt, M.W., Schmidt, K., Röthig, T.R., Chmiel, H., 1990. Rejection and
continuous regeneration of the native coenzyme NAD(P)H in a charged ultrafiltration membrane enzyme reactor. Ann. NY Acad. Sci. 613, 820–826.
Larsson, P.O., Mosbach, K., 1974. The preparation and characterisation of a watersoluble coenzymically active dextran-NAD+ . FEBS Lett. 46 (1), 119–122.
Le, M., Means, G.E., 1998. NAD+ /NADH recycling by coimmobilized lactate dehydrogenase and glutamate dehydrogenase. Enzyme Microb. Technol., 49–
57.
Lin, J., Siddiqui, J.A., Ottenbrite, R.M., 2001. Surface modification of inorganic oxide
particles with silane coupling agent and organic dyes. Polym. Adv. Technol. 12
(5), 285–292.
Lin, S.S., Harada, T., Hata, C., Miyawaki, O., Nakamura, K., 1997. Nanofiltration membrane bioreactor for continuous asymmetric reduction of 2-ketoglutarate to
produce l-glutamate with NADH regeneration. J. Ferment. Bioeng. 83 (1), 54–
58.
Lin, S.S., Miyawaki, O., Nakamura, K., 1999. Continuous production of l-carnitine with
NADH regeneration by a nanofiltration membrane reactor with coimmobilized
l-carnitine dehydrogenase and glucose dehydrogenase. J. Biosci. Bioeng. 87 (3),
361–364.
107
Liu, W., Wang, P., 2007. Cofactor regeneration for sustainable enzymatic biosynthesis.
Biotechnol. Adv. 25 (4), 369–384.
Månsson, M.O., Larsson, P.O., Mosbach, K., 1979. Recycling by a second enzyme of
NAD covalently bound to alcohol dehydrogenase. FEBS Lett. 98 (2), 309–313.
Nidetzky, B., Haltrich, D., Kulbe, K.D., 1996. Carry out coenzyme conversions economically. Chem. Tech. 26, 31–36.
Nidetzky, B., Schmidt, B., Neuhauser, W., Haltrich, D., Kulbe, K.D., 1994. Application of
charged ultrafiltration membranes in continuous, enzyme-catalyzed processes
with coenzyme regeneration. In: Pyle, D.L. (Ed.), Separation for Biotechnology.
Royal Society of Chemistry, Cambridge, pp. 351–357.
Obón, J.M., Almagro, M.J., Manjón, A., Iborra, J.L., 1996. Continuous retention of
native NADP(H) in an enzyme membrane reactor for gluconate and glutamate
production. J. Biotechnol. 50 (1), 27–36.
Obón, J.M., Manjón, A., Iborra, J.L., 1998. Retention and regeneration of native NAD(H)
in noncharged ultrafiltration membrane reactors: application to l-lactate and
gluconate production. Biotechnol. Bioeng. 57 (5), 510–517.
Röthig, T.R., Kulbe, K.D., Bückmann, F., Carrea, G., 1990. Continuous coenzyme dependent stereoselective synthesis of sulcatol by alcohol dehydrogenase. Biotechnol.
Lett. 12 (5), 353–356.
Stengelin, M., Patel, R.N., 2000. Phenylalanine dehydrogenase catalyzed reductive
amination of 6-(1 ,3 -dioxolan-2 -yl)-2-keto-hexanoic acid to 6-(1 ,3 -dioxolan2 -yl)-2S-aminohexanoic acid with NADH regeneration and enzyme and
cofactor retention. Biocatal. Biotransform. 18 (5), 373–400.
Sundberg, L., Porath, J., 1974. Preparation of adsorbents for biospecific affinity chromatography. I. Attachment of group-containing ligands to insoluble polymers
by means of bifunctional oxiranes. J. Chromatogr. 90 (1), 87–98.
Wandrey, C., Bossow, B., 1986. Continuous cofactor regeneration-utilization of polymer bound NAD(H) for the production of optically active acids. Biotechnol.
Bioind. 3, 8–13.
Wang, P., 2006. Nanoscale biocatalyst systems. Curr. Opin. Biotechnol. 17 (6),
574–579.
Wykes, J.R., Dunnill, P., Lilly, M.D., 1975. Cofactor recycling in an enzyme reactor. A
comparison using free and immobilized dehydrogenases with free and immobilized NAD. Biotechnol. Bioeng. 17, 51–68.
Yamazaki, Y., Maeda, H., 1982. The co-immobilization of NAD and dehydrogenases
and its application to bioreactors for synthesis and analysis. Agric. Biol. Chem.
46 (6), 1571–1581.
Yamazaki, Y., Maeda, H., Suzuki, H., 1976. Application of polylysine bound succinylNA to a membrane reactor. Biotechnol. Bioeng. 18 (12), 1761–1775.
Yokokawa, R., Tarhan, M.C., Kon, T., Fujita, H., 2008. Simultaneous and bidirectional
transport of kinesin-coated microspheres and dynein-coated microspheres on
polarity-oriented microtubules. Biotechnol. Bioeng. 101 (1), 1–8.
Yu, Y.T., Chang, T.M.S., 1981a. Lipid-polymer membrane artificial cells containing.
Multienzyme systems, cofactors and substrates for the removal of ammonia and
urea. Trans. Am. Soc. Artif. Intern. Organs 27, 535–538.
Yu, Y.T., Chang, T.M.S., 1981b. Ultrathin lipid-polymer membrane microcapsules containing multienzymes, cofactors and substrates for multistep enzyme reactions.
FEBS Lett. 125 (1), 94–96.
Yu, Y.T., Chang, T.M.S., 1982. Immobilization of multienzymes and cofactors within
lipid-polyamide membrane microcapsules for the multistep conversion of
lipophilic and lipophobic substrates. Enzyme Microb. Technol. 4, 327–331.
Zappelli, P., Rossodivita, A., Prosperi, G., Pappa, R., Re, L., 1976. New coenzymicallyactive soluble and insoluble macromolecular NAD+ derivatives. Eur. J. Biochem.
62, 211–215.
Zappelli, P., Rossodivita, A., Re, L., 1975. Synthesis of coenzymically active soluble and
insoluble macromolecularized NAD+ derivatives. Eur. J. Biochem. 54, 475–482.