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Digital Object Identifier: http://dx.doi.org/10.4172/2329-6674.1000117
Sinha et al., Enz Eng 2013, 2:2
http://dx.doi.org/10.4172/2329-6674.1000117
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NAD (H) Linked Enzyme Catalyzed Reactions using Coupled Enzymes in a
Composite Nanoparticle System
Sujata Sinha*#, Vidya Bhat# and Subhash Chand
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi Hauz Khas, New Delhi, India
Abstract
Different approaches for cofactor recycling/regeneration have been studied and one of them is having
regenerating enzyme immobilized on some suitable support. Here, we are reporting the cofactor NAD (H) recycling
with the help of free enzymes and enzymes loaded on nanoparticles. Baker’s yeast alcohol dehydrogenase (ADH)
and formate dehydrogenase (FDH) from Candida boidinii were immobilized on alumina nanoparticles and applied
to catalyze the coupled reactions for production of n-propanol. Cofactor regeneration within the reaction cycle was
achieved as a result of collision between enzyme loaded particles and free cofactor. Brownian motion provided
effective interactions among the catalytic components, and thus realized a dynamic shuttling of the cofactor between
the two enzymes to keep the reaction cycles continuing. Maximum recycle rate of 6650 cycles/hr was obtained and it
decreased with increasing cofactor concentration within the reaction system, for free as well as immobilized system.
It was concluded that particles attached enzymes could be one of the new biochemical strategy used for cofactor
dependent biotransformation.
Keywords: Immobilization; Nanoparticles; Cofactor regeneration;
catalyze coupled reactions for production of α-ketoglutarate and lactate
with cofactor regenerated within the reaction cycle [3]
Introduction
Other similar studies for cofactor recycling have been done by
Zahab et al. [6] and Luo et al. [7]. In the present work two enzymes
alcohol dehydrogenase (ADH) from Baker’s yeast and formate
dehydrogenase (FDH) from Candida boidinii were immobilized on
Alumina nanoparticles and cofactor recycling reactions were carried
out both by free and immobilized enzyme systems and efforts were made
to develop an efficient cofactor recycling system. It was assumed that
ADH will catalyze reaction propionaldehyde to n-propanol generating
reduced form of NAD i.e., NADH which in turn will be oxidized by
FDH using Sodium formate as substrate. Product of these reactions of
Sodium formate will generate water and carbon dioxide which are non
interfering with the reaction set up and can easily be removed.
TTN
Cofactors dependent Oxidoreductase enzymes are of great
industrial use in biotransformation reaction [1,2]. A majority of these
enzymes require coenzymes such as NAD (H), NADP (H) and ATP
for their actions. In recent years, NAD (H) and NADP (H) based
reactions have been examined extensively for their chemical processing
applications [3]. Because of the stoichiometric requirement and
prohibitively high costs of NAD (H), cofactor-linked enzymes have not
found to be of much use at commercial scale. Efficient regeneration and
reuse of cofactors are essential for large scale synthetic applications of
cofactor based enzymes [4]. Therefore, to maintain cost effectiveness in
the use of cofactor-based enzymes at commercial scales, it is necessary
to develop an efficient method to recycle them in situ. In addition,
cofactor regeneration can also drive the reaction to completion,
simplify product isolation, and allow the removal of inhibitory cofactor
byproducts, further reducing the cost of synthesis.
Chemical, electrochemical, photochemical, microbial and
enzymatic reactions have all been developed for cofactor regeneration
[4]. Enzymatic method is preferable due to its high efficiency and
selectivity. There are two ways to achieve the enzymatic regeneration;
one is through the use of substrate coupled reaction system and the
second is through the use of second enzyme for recycling of cofactor.
For majority of cofactor regeneration reaction, the use of second
enzyme is preferred [3] because of its affordability of broader option of
substrates. Cofactor regeneration with the help of immobilized enzyme
systems which is preferred in case of industrial systems for recovery
and reuse of enzyme is desirable.
Solid support attached insoluble cofactor and enzymes are much
easier to reuse and may afford more flexible reactor design as compared
to membrane systems.
However, most of the immobilized cofactors have been used with
free enzymes, as it was difficult to achieve activity with systems that
have cofactor and enzyme both immobilized [5]. In one of the study,
silica nanoparticles supported enzymes: Lactate Dehydrogenase (LDH);
Glutamate Dehydrogenase (GLDH) and NAD (H) were prepared to
Enz Eng
ISSN: 2329-6674 EEG, an open access journal
Materials and Methods
Materials
Alcohol dehydrogenase (ADH) (EC 1.1.1.1, lyophilized powder)
from Baker’s yeast, NAD (H), NAD, propionaldehyde and n-propanol
were all purchased from SRL (Sisco Research Lab Private Ltd.
Mumbai, India). Formate dehydrogenase (FDH) from C.boidinii
(EC:1.2.1.2,lyophilized powder), 3-aminopropyl Trimethoxy silane
(APS) and Bovine serum albumin (BSA) were purchased from Sigma
Chemical Co. (St. Louis, USA) Sodium Formate was purchased from
*Corresponding author: Sujata Sinha, Department of Biochemical Engineering
and Biotechnology, Indian Institute of Technology, Delhi Hauz Khas, New
Delhi-110016, India, Tel: +91-26591004; Fax :+91-1126582282; E-mail:
[email protected]
Received November 11, 2013; Accepted December 03, 2013; Published
December 06, 2013
Citation: Sinha S, Bhat V, Chand S (2013) NAD (H) Linked Enzyme Catalyzed
Reactions using Coupled Enzymes in a Composite Nanoparticle System. Enz Eng
2: 117. doi:10.4172/2329-6674.1000117
Copyright: © 2013 Sinha S, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Volume 2 • Issue 2 • 1000117
Citation: Sinha S, Bhat V, Chand S (2013) NAD (H) Linked Enzyme Catalyzed Reactions using Coupled Enzymes in a Composite Nanoparticle
System. Enz Eng 2: 117. doi:10.4172/2329-6674.1000117
Page 2 of 5
Hi Media Laboratories Pvt. Ltd. Mumbai, India. Glutaraldehyde
was procured from Loba Chemie Pvt. Ltd., Bombay. Alumina and
Zirconium nanoparticles were a kind gift from CGCRI (Centre for glass
and Ceramics Research Institute, Kolkata, India).
Functionalization of nanoparticles
Prefabricated alumina nanoparticles were functionalized using
two different methods, modified procedures for silanization of
alumina sheets [8] and silica gel [9] respectively. 0.5 g of dry alumina
nanoparticles were silanized using 10 ml of 5% APS (Aminopropyl
trimethoxysilane) in dry acetone for 1 h (at RT and neutral pH). The
nanoparticles were recovered by centrifugation at 8000 rpm for 30 min.
Excess silane was washed off with acetone and the nanoparticles baked
at 100°C overnight in an oven. The dried nanoparticles were then treated
with 2.5% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.05) for one
hour, washed and stored as a wet cake till further use (method 1). In
another method (method 2) same process was modified by treating dry
nanoparticles with 10 ml of 10% aqueous APS (pH 3.45) at 75°C for 2.75
hours in a temperature controlled water bath and washing was done
with distilled water. In case of prefabricated zirconium nanoparticles,
same methods were performed as that followed by Chen et al. [10].
Enzyme immobilization
Immobilization of enzyme on alumina nanoparticles were carried
both with adsorption method and by covalent coupling. Physical
adsorption of enzymes on alumina nanoparticles (not functionalized
before use) were checked for suitability as a viable method for
immobilization by allowing suitably diluted enzyme (1 mg/ml, 0.1
M phosphate buffer, pH 7.05) to react with alumina nanoparticles at
5-10°C for 2 hours. The nanoparticles were collected by centrifugation
at 8000 rpm for 30 minutes and the supernatant stored for checking the
presence of unbound enzyme. The particles were washed and stored.
Covalent coupling was performed by same method except that particles
used were functionalized by above mentioned Method 1 and 2. In case
of zirconium, functionalized nanoparticles were added to phosphate
buffer (10 ml) of same pH as that of enzyme solution, immobilized
solid particles were collected by centrifugation, followed by repeated
washing with buffer solution and stored after freeze drying.
Activity assay
ADH activity was measured by monitoring the decrease in NADH
absorbance at 340 nm with time. The final reaction mixture consisted of
30 mM propanaldehyde, 0.3 mM NADH and suitably diluted enzyme
solution in 0.1 M phosphate buffer (pH 7.05) containing 0.1% BSA. The
reaction was started by the addition of 100 μl of the suitably diluted
enzyme and the absorbance at 340 nm was monitored for 5 minutes.
One Unit of enzyme activity was defined as enzyme required for oxidize
one micromole of NADH/minute. FDH activity was measured by
monitoring the increase in absorbance at 340 nm due to the generation
of NADH. One unit of FDH activity was defined as the formation of
1.0 µmol of NADH per min under the standard assay conditions (pH
7.4, 25°C). The activities of enzyme loaded particles were measured
in a batch reactor. Suitably diluted nanoparticles conjugated with
enzyme suspensions were prepared and added to the reaction mixture
containing respective substrates and cofactors, as the case may be.
Separate blanks were used for each sample containing a particular
amount/concentration of nanoparticles. Simultaneously, enzyme
activity was also determined in the supernatant.
Enzyme loading and specific activity
Different amounts of enzymes (5, 10 and 20 mg) were incubated
Enz Eng
ISSN: 2329-6674 EEG, an open access journal
with the same amount of nanoparticles to determine the loading
capacity of the particles. Enzyme loading was calculated as activity
units retained/ amount of nanoparticles. Further, the protein loading
was checked to ascertain the changes (if any) in the specific activity of
the enzyme upon immobilization. The protein loading was determined
based on the difference of the protein content in solution (including
wash) before and after immobilization, by Folin-Lowry [11] method.
Characterization of immobilized enzymes on nanoparticles
Nanoparticulate systems morphology such as shape and occurrence
of aggregation phenomena was studied by scanning electron microscopy
(SEM). Samples of nanoparticles were mounted on metal stubs, gold
coated under vacuum and then examined on a Carl Zeiss EVO Series
Scanning Electron Microscope.
Effect of pH on immobilization of enzyme:
Enzyme and metal nanoparticle solution was prepared in buffer of
different pH (4.0, 9.0, 7.5) and same procedure of immobilization was
repeated in all the cases.
Reusability and stability assay
Reusability assay of enzyme immobilized nanoparticles were done
by repeated washing with phosphate buffer before adding to second set
of reactions. Stabilities of free and immobilized enzymes were checked
at different time interval in same storage condition and compared.
Coupled Enzymatic Reactions
Coupled enzymatic reactions were set up using enzymes (ADH &
FDH), substrates (propanaldehyde and Sodium formate), cofactors and
analysis was done both by spectrophotometer (Optizen 3220 UVbio) and
by Gas chromatography (HP Nucon, 6500). Effect of changing enzyme
activity ratios on the steady state cofactor concentration was studied
by using a constant ADH concentration and the FDH concentrations
were suitably adjusted to get final ADH to FDH activity ratios of 2.5,
5 and 10.28. The final substrate concentrations in the reaction cocktail
consisted of 30 mM propanaldehyde, 30 mM sodium formate and 0.3
mM NADH. Spectrophotometrically, at time zero, ADH was added and
the reaction was monitored for 5 minutes by measuring the absorbance
at 340 nm. Then, the addition of FDH was carried out and the coupled
reaction was monitored for another 10 minutes (till steady state was
reached w.r.t the cofactor concentrations in the reaction). Combined
reactions were also monitored by gas chromatography; three different
systems were tried, containing both free enzymes, one immobilized
enzyme (ADH) and both enzymes immobilized respectively. The
reactions were carried out in 0.1 M potassium phosphate buffer (pH
7.5) at room temperature with magnetic stirring, with the amount of
each catalytic component controlled to achieve the desired molar ratio.
A typical reaction included addition of 3 mg ADH-carrying particles
and 30 mg FDH-carrying particles in 1mL reaction mixture containing
0.8 M propanaldehyde, 0.8 M sodium formate and 1 mM NADH. 50
μl aliquots were collected and centrifuged to remove the immobilized
enzymes, and the supernatants used for GC analysis. The reactions were
monitored by measuring the rate of disappearance of substrate which is
propanaldehyde in the combined reaction. Controls without enzymes
were used to check for loss of propanaldehyde due to other factors.
Results & Discussion
Enzyme immobilization on nanoparticles and mechanisms
Adsorption method of immobilization was ineffective in case
Volume 2 • Issue 2 • 1000117
Citation: Sinha S, Bhat V, Chand S (2013) NAD (H) Linked Enzyme Catalyzed Reactions using Coupled Enzymes in a Composite Nanoparticle
System. Enz Eng 2: 117. doi:10.4172/2329-6674.1000117
Page 3 of 5
of binding of enzymes to alumina nanoparticles while it worked for
zirconium nanoparticles. No detectable activity was observed in either
the supernatant or in the particles by adsorption method when enzymes
were added to alumina. This suggests that direct contact with the surface
of the alumina particles denatures the enzyme completely. Unsuccessful
direct coupling of the enzyme with the alumina nanoparticles suggested
the need for surface modification of the particles by molecules of
desired properties. Biofunctionalization of metals with the use of
silane precursors involves treating the surface hydroxyl groups with
trialkoxyaminosilanes and consequent functionalization using different
routes for modification of the primary amino groups. Modification
is carried out for covalent coupling of enzymes with the help of
glutaraldehyde, a bi-functional molecule with two aldehyde groups.
Upon reaction, one of the aldehyde groups reacts with the amino group
on the aminosilanes to form an amide bond, leaving the other aldehyde
group free to react with the secondary amino groups present on the
amino acids comprising the protein (Figure 1). Nearly equal activity,
equivalent to 4.8 mg of the free enzyme, was retained in the supernatant
in both methods of alumina functionalization. However, the enzyme
loading on the particles varied (Figures 2 and 3). In case of zirconium,
7.5 pH, room temperature (28°C), contact time of 24 hours and enzyme
concentration 1.2 mg/ml were considered optimum for immobilization.
A typical SEM micrograph for the metal nanoparticles without and
with bound enzymes has been shown in Figure 4a and b. Nanoparticle
aggregates were clearly visible and had a mean diameter of 250 nm,
after binding enzymes their mean diameter increases slightly almost
similar to that of unbound ones, this reveals that binding process did
not significantly result in change in size of particles this could be due
to change occurred only on particles surface, same case was reported
by Li et al. [12] in case of binding of SCAD on magnetic nanoparticles.
Figure 2: Immobilized enzyme activity assays. M1: Method 1; M2: Method 2;
Sup: Supernatant; Particles: Nanoparticle suspensions. The sample dilutions
are also mentioned.
Enzyme loading and specific activity
Alumina particles functionalized by Method 1 showed retention
of 470 units of enzyme activity whereas the corresponding value for
nanoparticles prepared by Method 2 was 14.44 units and enzyme
loading in Method 1 was 11.77 U (mg particles) as compared to 0.35
in Method 2. It was nearly 30 times more than that in Method 2.
This could be attributed to the limited stability of covalently bonded
molecules on alumina due to the activation of the Al-O-Si bond
Figure 3: Activity retention on particles and supernatant post Immobilization.
(a)Alumina
(a) Zirconium
Figure 1: Schematic representation of the process of biofunctionalization of
alumina nanoparticles.
Enz Eng
ISSN: 2329-6674 EEG, an open access journal
(b) Alumina with enzyme
(b) Zirconium with enzyme
Figure 4: Scanning electron micrographs of metallic nanoparticles (a)
Alumina and Zirconium
without enzymes (b)
Alumina and Zirconium with bound enzymes.
Volume 2 • Issue 2 • 1000117
Citation: Sinha S, Bhat V, Chand S (2013) NAD (H) Linked Enzyme Catalyzed Reactions using Coupled Enzymes in a Composite Nanoparticle
System. Enz Eng 2: 117. doi:10.4172/2329-6674.1000117
Page 4 of 5
breakage due to a local increase in pH near the surface. This increase
in pH has been attributed to the presence of amino groups in the APS
molecule, and becomes significant when the particles are exposed to
aqueous solutions before the conversion of the amine groups to amides
by glutaraldehyde. Silanization in Method 1 was carried out in dry
acetone, whereas Method 2 used aqueous solutions. Hence, the lower
loading in Method 2 can be attributed to the breakage of the Al-OSi bond, resulting in lesser glutaraldehyde molecules available at the
surface for coupling. Therefore, Method 1 was chosen as the method
of choice for immobilization of ADH/FDH on alumina nanoparticles.
The maximum loading of 10.7 ± 0.5 U/mg was achieved with 10 mg of
ADH and hence this amount was used in all further experiments. For
coupling of FDH, using an initial amount of 10 mg FDH, a loading of
0.077 U/mg was achieved. However, optimization could not be carried
out due to limited quantity of FDH available and the above preparation
was used for the experiments. The protein loading on the nanoparticles
was determined as described previously to assess the changes in specific
activity of the enzyme upon immobilization on nanoparticle supports.
The protein content of the commercial enzyme preparation for ADH
was about 30% (i.e., 0.3 mg protein/mg solid) while that for FDH was
43%. An increase in the specific activity (Vmax) for the enzymes is
obtained upon the immobilization of the enzymes upon nanoparticle
supports. Such a trend has been seen in other studies as well [13]. The
reason for this behavior is not clear. However, in the aforementioned
study; a corresponding decrease in activation energy for the enzyme
was noted upon immobilization, which may be responsible for the
increase in activity. In case of zirconium loading of enzyme (ADH) was
calculated as 1.0 U/mg of nanoparticles.
Figure 5: Effect of enzyme activity ratio on steady state cofactor
concentrations.
Stability of immobilized enzyme
The free enzyme (ADH) solutions lost 24 and 72% of their activities
upon storage at 4-8°C for 24 and 48 hours, respectively. In contrast, the
immobilized enzymes lost about 3 and 25% of their activity after 24
hours and one week, respectively. Therefore, the stability of the enzymes
increased due to their covalent coupling with the functionalized alumina
particles. The reutilization capacity of the immobilized enzymes on
zirconium particles were up to three cycles with minor loss of activity.
Influence of pH during enzyme immobilization
It has been reported that the pH value of the buffer solution during
immobilization has a great influence on the activity and enantioselectivity
of the biocatalyst [14]. In this work, the immobilization of enzyme
(ADH) on zirconium was carried out at pH 4.0, 5.0, 7.5 and 9.0. Similar
enzyme loadings within the range of 0.0040-0.0095 mg/mg of matrix
are obtained, but the activities are quite different with optimal results
achieved at pH values of 7.5.
Coupled enzyme reactions
Effect of ratio of enzyme activities on the steady state cofactor
concentrations: Addition of second enzyme (FDH), led to gradual
increase in absorbance due to production of NADH by FDH, with
the absorbances indicating the attainment of steady state within 10
minutes (Figure 5). Increasing FDH concentrations (or decreasing the
ADH/FDH activity ratios) leads to higher steady state concentrations
of NADH. For example, when ADH/FDH activity ratio=10.28, nearly
88% of the NADH added initially is in the form of its oxidized form
NAD+ at steady state (Figure 6). Therefore, we can control the steady
state concentrations of the two forms of the cofactor (oxidized and
reduced) by altering the activities of the enzymes in the reactions. Since
the reduced form of the NAD (H) cofactor is less stable in solution,
Enz Eng
ISSN: 2329-6674 EEG, an open access journal
Figure 6: Fraction of cofactor existing as NADH at steady state as a function
of the enzyme activity ratio.
in subsequent experiments, high ADH/FDH ratios were used to retain
greater percentage of the cofactor in its oxidized form.
Total Turnover Number and Cofactor recycling rate: Coupled
reactions were first carried out with free enzymes as that of Wykes
et al. [5] in 250 μl reaction volumes in ELISA plates with continuous
shaking. Using 0.8 M of substrates, 21.54 and 2.09 units of ADH and
FDH respectively (ADH/FDH=10.28) and different concentrations
of the cofactor, the reaction was carried out for 1 hour. A maximum
Total Turnover Number (TTN=moles of product formed/moles of
cofactor added) of 1320 was achieved. The cofactor recycling rate,
i.e., the moles of product formed per hour normalized by the moles
of cofactor added, was same as the TTN in this case (Table 1). When
similar experiments were carried out with free FDH and immobilized
ADH added in the same activity ratios, results were similar to the free
enzyme systems, indicating that such systems perform equally well.
Further, the examination of TTN in systems containing both enzymes
immobilized indicated similar dynamic cofactor-enzyme interactions.
Using 0.8 M substrates, 0.5 mg ADH loaded particles and 7.5 mg
FDH loaded particles, the reaction was carried out for 5 hours and a
maximum TTN of 2900 was reached (equivalent to 580 h-1) with the
lowest concentration of cofactor used being 140 μM (Table 2). Also,
increasing the cofactor concentration lowered the TTN by the same
Volume 2 • Issue 2 • 1000117
Citation: Sinha S, Bhat V, Chand S (2013) NAD (H) Linked Enzyme Catalyzed Reactions using Coupled Enzymes in a Composite Nanoparticle
System. Enz Eng 2: 117. doi:10.4172/2329-6674.1000117
Page 5 of 5
[NADH] (mM)
Conclusion
TTN/Cofactor recycling rate (h-1)
0.3
1320
1.0
400
1.42
270
5.0
80
Table 1: TTN and Cofactor Recycling Rate (CRR) at different initial cofactor
concentrations for coupled reactions with free enzymes; CRR was calculated as
number of moles of product formed per hour per mole of cofactor added.
factor
recycling rate(h-1)
0.14
2900
0.28
1500
300
0.57
690
138
1.14
330
66
580
Table 2: TTN and cofactor recycling rates at different initial cofactor concentrations
for coupled reactions with immobilized enzymes.
ratio, which can be attributed to the low Km values for NADH that
maximize the reaction rates at very low concentrations of the cofactor.
Cofactor recycling experiments were not carried out by zirconium
immobilized enzymes due to lack of resources and time.
In cofactor regeneration experiments, cofactor recycling rate is a
very important parameter since it can affect the recycling capacity and
hence the TTN achievable by the system. Higher cofactor recycle rates
can be achieved by increasing the concentration of the substrates and/
or the amount of enzymes.
Effect of substrate concentration on cofactor recycle: The substrate
concentrations are expected to affect the TTNs by affecting the rate of
reaction of the enzyme. Two different concentrations of propanaldehyde
(0.5 and 0.8 M) were tested and no difference was found in the TTNs,
with the absolute molar conversion of propanaldehyde being similar
at comparable time points (the total reaction time for the smaller
concentration being proportionally smaller). These concentrations were
well above the Km for propanaldehyde, however, the effect of lowering
the concentration of propanaldehyde below its Km value could not be
determined, because of the concentration range in contention being
well below the limit of detection by gas chromatography.
Effect of enzyme concentration on cofactor recycle rates:
Increasing the amount of enzymes in the system can improve the
recycle rates as depicted above. A maximum rate of 6650 cycles/hour
was achieved with 0.14 mM cofactor and the highest amounts of
enzymes used in the experiment. Increasing the amount of enzymes
may further increase the recycle rate; however, care has to be taken
to maintain a high liquid-to-solid ratio in the system. This is because
particle mobility is an important criterion in determining the efficiency
of cofactor recycling in such systems. Higher particle concentrations
are required to improve the collision frequency of the particles, which
would facilitate the reactions. However, at higher concentrations,
aggregation of the particles may be a problem. Therefore, a balance
between the two is necessary, and hence it is expected that below a
certain liquid-to-solid (w/w) ratio, increasing the amount of particles
(consequently the enzymes) would result in a decrease in the cofactor
recycle rates. The recycle rates can be further improved by optimizing
the factors influencing the performance of the enzymes in the system
- the buffer composition, its pH, the immobilization system and by
increasing the stability of the cofactor in the system - by controlling
the form of the cofactor at steady state and/or by immobilization of the
cofactor itself.
Enz Eng
ISSN: 2329-6674 EEG, an open access journal
ADH and FDH enzymes were immobilized on metals nanoparticles
(i.e., Alumina and Zirconium) and a cofactor recycling system was
developed consisting of both enzymes immobilized separately on
Alumina nanoparticles and free cofactors. As expected, mobility of
particles is critical to realizing dynamic enzyme–cofactor interactions,
and efficient recycling of cofactor 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
Financial support from Lockheed Martin Corporation USA is gratefully
acknowledged. We thank CGCRI Kolkata for providing nano particles and Dr
Sanjay Dhakate, NPL for SEM and TEM facility for nanoparticle characterization
during the study.
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Citation: Sinha S, Bhat V, Chand S (2013) NAD (H) Linked Enzyme Catalyzed
Reactions using Coupled Enzymes in a Composite Nanoparticle System. Enz
Eng 2: 117. doi:10.4172/2329-6674.1000117
Volume 2 • Issue 2 • 1000117