Catalysis Communications 53 (2014) 62–66

Contents lists available at ScienceDirect

Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom

Short Communication

Preparation of stable cross-linked enzyme aggregates (CLEAs) of

NADH-dependent nitrate reductase and its use for silver nanoparticle
synthesis from silver nitrate
Sachin Talekar a,⁎, Gandhali Joshi a, Radhika Chougle a, Basavaraj Nainegali a, Shashikant Desai b, Asavari Joshi a,
Shashikant Kambale a, Priyanka Kamat a, Rutumbara Haripurkar a, Suraj Jadhav a, Shamraja Nadar a
a
Department of Biotechnology Engineering, Kolhapur Institute of Technology's College of Engineering, Kolhapur 416 234, India
b
Department of Chemical Engineering, Tatyasaheb Kore Institute of Engineering and Technology, Warananagar, Kolhapur 416113, India

a r t i c l e i n f o a b s t r a c t

Article history: The NADH-dependent nitrate reductase from Fusarium oxysporum cell extract was directly immobilized as cross
Received 20 March 2014 linked enzyme aggregates (CLEAs) and investigated for the synthesis of silver nanoparticles by a reduction of
Received in revised form 9 May 2014 silver nitrate. The effects of precipitant type and cross-linking on activity recovery of enzyme in CLEAs were
Accepted 10 May 2014
studied. After aggregation of enzyme with ammonium sulfate followed by cross-linking formed aggregates for
Available online 16 May 2014
4 h with 8 mM glutaraldehyde, 93% activity recovery was achieved in CLEAs with enhanced thermal stability
Keywords:
at 50 °C and 40 °C. Scanning electron microscopy analysis showed that immobilized NADH-dependent nitrate
Enzyme catalysis reductase was of spherical structure. CLEAs showed 90% catalytic yield even after 4 cycles of repeated use in silver
NADH-dependent nitrate reductase nanoparticle synthesis at pH 7.2 and temperature 35 °C.
Enzyme immobilization © 2014 Elsevier B.V. All rights reserved.
Cross-linked enzyme aggregates
Biosynthesis of metal nanoparticles
Silver nanoparticles

1. Introduction
In the context of synthetic chemistry, in vitro enzyme catalyzed reac-
tions are now widely recognized as practical alternatives to traditional
(non-biological) organic synthesis of complex natural products and
small molecules [1]. By contrast, the use of in vitro enzymatic reactions
for synthesis of inorganic nanoparticles is relatively rare and very
much under-utilized. Silver nanoparticles are one of the most commer-
cialized inorganic nanomaterials. To design a rational enzymatic strate-
gy for the synthesis of silver nanoparticles on large scale, Kumar et al. [2]
purified NADH-dependent nitrate reductase from Fusarium oxysporum
cell extract and used the pure enzyme for in vitro biosynthesis of silver
nanoparticles. But the commercial viability of industrial biosynthesis
stands or falls with the cost contribution of the enzyme. The use of
purified enzymes in soluble form leads to not only the waste but also
the limited reuse of enzymes. Additionally, soluble enzymes when
used remain as impurity in nanoparticle. When immobilized enzymes
are used this can be ruled out where the enzymes can be reused and
nanoparticle's downstream steps can be very much reduced. This
⁎ Corresponding author at: Department of Biotechnology Engineering, Kolhapur
Institute of Technology's College of Engineering, Gokul Shirgaon, Kolhapur 416 234,
Maharashtra, India. Tel.: +91 0231 2638141; fax: +91 0231 2638881.
E-mail address: sachintalekar7@gmail.com (S. Talekar).
would do away the downstream processing required for the use of silver
nanoparticles in homogeneous catalysis and non-linear optics [3].
Compared with carrier-bound immobilization techniques, cross-
linked enzyme aggregates (CLEAs) have a prominent advantage of
high volumetric activity and space time yield since the activity dilution
caused by solid carriers is avoided [4]. Principally, CLEAs are prepared by
precipitating the enzyme by the addition of a salt or an organic solvent
to soluble enzyme followed by cross-linking of the resulting enzyme ag-
gregates with glutaraldehyde. Glutaraldehyde, a bi-functional reactive
agent, is capable of cross-linking enzyme molecules in aggregates via
the reaction of surface amine residues of enzyme to yield insoluble
biocatalysts with high activity. Since precipitation is often used to purify
enzymes, enzyme immobilization as CLEAs combines purification and
immobilization into single unit operation. Compared to immobilization
using porous, non-porous and hetero-functional supports, enzyme im-
mobilization using CLEA technology is compatible with most contami-
nant proteins, because in any case the target enzyme may precipitate
under the appropriate conditions specific for each enzyme [5]. There-
fore it is possible to isolate an enzyme in its immobilized form CLEAs di-
rectly from a crude extract [6]. In many reports CLEAs have been
successfully prepared from crude enzyme extracts [7–10]. Moreover,
due to cross-linking of subunits, immobilization of enzymes as CLEAs
stabilizes the quaternary structure of multimeric enzymes even better
than multi-subunit covalent attachment in preexisting supports

http://dx.doi.org/10.1016/j.catcom.2014.05.003
1566-7367/© 2014 Elsevier B.V. All rights reserved.
 S. Talekar et al. / Catalysis Communications 53 (2014) 62–66 63

preventing their dissociation and loss of activity of multimeric enzymes
[11–13]. As NADH dependent nitrate reductase is dimeric enzyme, it is
therefore beneficial to immobilize it as CLEAs to hold the catalytically
active dimer together.
No studies have been reported on CLEAs of NADH-dependent ni-
trate reductase and no attempts have been made to use CLEAs of
NADH-dependent nitrate reductase as immobilized biocatalyst for
biosynthesis of silver nanoparticles. Therefore, herein, we report
the work from this nascent field, with its beginnings with the
preparation of NADH-dependent nitrate reductase CLEAs from
F. oxysporum cell extract and then the demonstration of synthesis
of silver nanoparticles by the reduction of silver nitrate using
NADH-dependent nitrate reductase CLEAs. The primary reaction in-
volved the electron transfer from NADH to silver nitrate catalyzed
by NADH-dependent nitrate reductase CLEAs as immobilized biocat-
alyst. In this reaction 8-hydroxyquinoline was used as electron shut-
tle transferring the electron generated during reduction of nitrate to
silver ions. Due to the large size of CLEA particles than nanoparticles,
CLEA particles could be easily separated from reaction mixture by
centrifugation at low speed or simple filtration.

2. Experimental

2.1. Preparation of NADH-dependent nitrate reductase CLEAs

2.1.1. Precipitation of enzyme

Chilled organic solvents (acetone, n-propanol, ethanol, acetonitrile, DMSO, 10 mL each) and saturated ammonium sulfate solution (10 mL) were
added drop wise separately to the samples of cell free filtrate (5 mL containing 35 U of NADH-dependent nitrate reductase and protein content 2 mg/mL)
with shaking and kept for 30 min at 4 °C for complete precipitation of enzyme and then centrifuged for 10 min at 10,000 ×g. The precipitates and
supernatants were collected separately. The precipitate was redissolved in 25 mM sodium phosphate buffer, pH 7.2. The NADH-dependent nitrate
reductase activity was then checked in both supernatants and aliquots of redissolved precipitate.

2.1.2. Cross-linking of enzyme

Saturated ammonium sulfate solution (10 mL) was added to cell free filtrate (5 mL containing 35 U of NADH-dependent nitrate reductase and
protein content 2 mg/mL) in capped centrifuge tube. After keeping the mixture for 30 min at 4 °C for complete precipitation of enzyme, glutaralde-
hyde was added to a final concentration of 8 mM. The mixture was kept at 30 °C for 4 h with constant shaking at 150 rpm. Then the suspension was
centrifuged at 10,000 × g for 10 min. The supernatant was collected and checked for NADH-dependent nitrate reductase activity. The pellet was
washed three times with 25 mM sodium phosphate buffer, pH 7.2 to remove unreacted glutaraldehyde and unbound proteins. The final enzyme
preparation was kept in the same buffer (1 mL) at 4 °C. The percent activity recovery of NADH-dependent nitrate reductase in CLEAs was determined
using following equation:

Activity recoveryð%Þ
Total activity of NADH−dependent nitrate reductase in CLEAsðUÞ
¼  100:
Total activity of NADH−dependent nitrate reductase used for CLEAs preparationðUÞ

2.2. Synthesis of silver nanoparticles using immobilized NADH-dependent nitrate reductase as CLEAs

Silver nanoparticles were synthesized by adding separately free enzyme and CLEAs to 10 ml reaction mixture prepared in 25 mM sodium phos-
phate buffer (pH 7.2) containing 1 mM AgNO3, 1 mM 8-hydroxyquinoline, 1 mM NADH and incubating at 35 °C with shaking (150 rpm). Equivalent
amount of NADH dependent nitrate reductase was taken in both free and CLEA form. Reactions were also performed in the absence of either the
NADH-dependent nitrate reductase CLEAs or NADH or 8-hydroxyquinoline separately. Aliquots of the reaction mixture were removed at intervals
and scanned in a double beam UV–vis spectrophotometer at 1 nm resolution.

2.3. Reusability of NADH- dependent nitrate reductase CLEAs

To evaluate the reusability of NADH-dependent nitrate reductase CLEAs for the synthesis of silver nanoparticles, CLEAs were subjected to silver
nanoparticle synthesis in batch mode at pH 7.2 and temperature at 35 °C. After a 24 h batch reaction, the CLEAs were separated by centrifugation,
washed twice with buffer and then suspended again in a fresh reaction mixture. At the end of each batch reaction cycle, the sample of reaction mix-
ture was scanned in a double beam UV–vis spectrophotometer at 1 nm resolution. The reduction efficiency of nitrate reductase was measured based
on relative absorbance at 426 nm calculated by taking the absorbance at 426 nm of the first batch reaction cycle as 100%.

3. Results and discussion
3.1. Preparation of NADH-dependent nitrate reductase CLEAs
Due to the different biochemical and structural properties of en-
zymes, the nature of best precipitant which precipitates maximum en-
zyme activity to be captured in CLEAs can vary from one enzyme to
another [14,15]. Therefore, we investigated five protein precipitating
agents: ammonium sulfate, acetone, ethanol, 2-propanol, n-butanol
and acetonitrile for evaluating their abilities of precipitating enzyme.
All examined organic solvents resulted in the total loss of enzyme
activity after precipitation. Only ammonium sulfate worked best,
precipitating 93% of soluble NADH-dependent nitrate reductase activity.
Therefore, ammonium sulfate was used as precipitating agent in further
experiments. The total loss of enzyme activity after precipitation with
organic solvents could be due to the hydrophobic interactions between
the solvent and the nonpolar groups of the NADH-dependent nitrate re-
ductase which shifts the equilibrium towards the denatured state [16].
It could also be explained due to the dissociation of subunits of NADH-
dependent nitrate reductase in organic solvent leading to complete in-
activation of enzyme [17].
The concentration of cross-linking agent must be optimized to re-
cover maximum enzyme activity in CLEAs. The effect of concentration
of cross-liking agent on the activity recovery of enzyme was studied
64 S. Talekar et al. / Catalysis Communications 53 (2014) 62–66

A linking occurred rapidly till 1 h of cross-linking reaction: after 1 h

cross-linking, almost 60% activity recovery was obtained in CLEAs
(Fig. 1B). Beyond 1 h, cross-linking continued but at a decreased rate
as activity recovery was increased from 60% to 93% till 4 h cross-linking.
This decreased rate of cross-linking might be due to an aldol condensa-
tion of free glutaraldehyde molecules with glutaraldehyde molecules
still grafted onto enzyme [20]. Cross-linking reactions with cross-
linking time greater than 4 h resulted in a decrease in the activity recov-
ery of enzyme in CLEAs but no enzyme activity was detected in superna-
tants collected after cross-linking times greater than 4 h. This result
implied that prolonged cross-linking time results in rigidification of en-
zyme due to a more intensive cross-linking which restricts enzyme flex-
ibility abolishing enzyme activity [21].

3.2. Structural characterization by scanning electron microscopy

Schoevaart et al. [22] have described that CLEAs have two types
based upon the morphology seen by scanning electron microscopy.
B Type 1 CLEAs appear as “spherical structures (balls)”, whereas type 2
CLEAs “cluster into less defined structures”. Due to clustering, type 2
CLEAs are course grained structures with fewer cavities compared to
fine grained structures of type 1 CLEAs with many cavities. Consequent-
ly, CLEAs with type 1 morphology aggregates permit better mass trans-
fer. As shown in scanning electron micrograph NADH-dependent
nitrate reductase CLEAs have spherical appearance (Fig. 2). Therefore,
NADH-dependent nitrate reductase CLEAs are of type 1 aggregate
form that might facilitate the movement of substrate to inner enzyme
molecules.

3.3. Thermal stability

The thermal stability of NADH-dependent nitrate reductase in free

enzyme and CLEAs were determined by incubating them in aqueous
buffer (pH 7.2) at different temperatures for different time intervals.
The residual activities were given in Fig. 3. At 50 °C, NADH-dependent
nitrate reductase in CLEAs retained 100% activity for at least 2 h of stor-
age time (Fig. 3A). As observed NADH-dependent nitrate reductase in
Fig. 1. Effect of glutaraldehyde concentration (A) and cross-linking time (B) on the activity CLEAs retained more than 70% of its initial activity after a 4 h incubation
recovery of NADH-dependent nitrate reductase in CLEAs. The 93% activity recovery corre-
sponds to 3.25 U/mg aggregate (total 32.5 U) of NADH-dependent nitrate reductase.
time at 50 °C and 40 °C, whereas residual activity of NADH-dependent
nitrate reductase in free enzyme was reduced to 15–35% in the same
conditions (Fig. 3A and B). These results indicated that the thermal sta-
using final glutaraldehyde concentration in the range of 2–50 mM. As bility of NADH-dependent nitrate reductase in CLEAs was much better
shown in Fig. 1A, the activity recovery of NADH-dependent nitrate re- than that of in free enzyme. This enhancement of CLEAs in thermal sta-
ductase in CLEAs was increased with an increase in glutaraldehyde con- bility might be due to intermolecular and intramolecular chemical
centration and 92.96% activity recovery was obtained at 8 mM cross-linking. Chemical cross-linking restricts any conformational
glutaraldehyde concentration. NADH-dependent nitrate reductase ac-
tivity was detected in supernatants collected after cross-linking using
glutaraldehyde concentrations less than 8 mM indicating insufficient
cross-linking at lower glutaraldehyde concentrations. No enzyme activ-
ity was detected in supernatants, and the activity recovery of enzyme
was decreased after cross-linking with glutaraldehyde concentrations
greater than 8 mM. It could be due to a drastic modification of enzyme
surface caused by uncontrolled polymerization of glutaraldehyde at
high concentration which results in a higher number of enzyme–
enzyme bonds per enzyme molecule lowering the catalytic activity of
enzyme [18]. Thus, it is convenient to activate the amino groups of the
enzyme with just one molecule of glutaraldehyde to get an intense
cross-linking [19]. Therefore, using 8 mM glutaraldehyde, one glutaral-
dehyde molecule per amino groups could have been introduced, and
this may be enough to cross-link the aggregates.
Cross-linking was carried out using 8 mM glutaraldehyde concentra-
tion supposing to introduce one glutaraldehyde molecule per amino
groups of enzyme. Therefore, there will not a real competition between
one-point modification and cross-linking which hinders the promotion
of cross-linking in the enzyme surface [18]. It was observed that cross- Fig. 2. Scanning electron micrograph of NADH-dependent nitrate reductase CLEAs.
 S. Talekar et al. / Catalysis Communications 53 (2014) 62–66 65

A
120
Free enzyme CLEAs

100
Residual activity (%)

80

60

40

20

0
0 1 2 3 4
Time (h)

B
120
Free enzyme CLEAs

100
Fig. 4. Transmission electron micrograph of silver nanoparticles synthesized with CLEAs.
Residual activity (%)

80
or 8-hydroxyquinoline was omitted from the reaction mixture, as
no surface plasmon absorption band at 426 nm was observed in reac-
60 tion mixtures. Fig. 4 shows the transmission electron microscope
(TEM) image of a typical sample of silver nanoparticles and indicates
40 the large quantity and good uniformity that were achieved using this
approach. These silver nanoparticles had a spherical shape and a size
in the range of 10–20 nm.
20

3.5. Reusability of NADH-dependent nitrate reductase CLEAs

0
0 1 2 3
Time (h)
Fig. 3. The thermal stability of NADH-dependent nitrate reductase in CLEAs and free
enzyme at (A) 50 °C and (B) 40 °C. The 100% residual activity of NADH-dependent nitrate
reductase corresponds to 32.5 U for CLEAs and 35 U for free enzyme.
change of the multimeric enzyme induced by heat and stabilizes the
quaternary structure of the enzyme [18].
3.4. Synthesis of silver nanoparticles using immobilized NADH-dependent
nitrate reductase as CLEAs
Formation of silver nanoparticles was followed by ultraviolet–visible
spectroscopy. Silver nanoparticles are known to exhibit a size-
dependent characteristic surface plasmon resonance band [23]. In the
case of free enzyme and CLEAs, a characteristic surface plasmon absorp-
tion band centered at 426 nm was observed in reaction mixture after
24 h of reaction (Fig. S1). The Fig. S2 shows the absorption of free enzyme
and CLEA reaction mixtures at 426 nm (Ag SPR) as a function of time. The
time course showed a sigmoidal shape which is characteristic of enzyme
catalysis reactions. Although in each case the silver nanoparticle synthesis
was started with an equivalent amount of NADH-dependent nitrate re-
ductase, it is observed that the silver nanoparticle synthesis performance
of the free form of enzyme was low compared to the immobilized form
CLEAs. Moreover, the rate of silver nanoparticle synthesis with CLEAs
was also high. The observed low silver nanoparticle synthesis perfor-
mance can be attributed to a lower thermal stability of free enzyme
compared to CLEAs. Silver nanoparticles did not form when either
NADH-dependent nitrate reductase in free and CLEA form or NADH
4
The reusability of CLEAs was studied up to four batch reaction cycles.
The UV–vis. absorption spectra of nanoparticles recorded from each
batch reaction cycle after 24 h showed single band absorption with
peak maximum at 426 nm without any shift in the peak (Fig. S3),
confirming the formation of nanoparticles of same morphology and di-
mensions in each cycle. A comparison of absorbance of peak maximum
at 426 nm obtained for each batch cycle shows that the recovered ni-
trate reductase CLEAs exhibit almost constant catalytic yield for at
least four successive cycles of the reduction reaction. In the fourth run,
the yield was still as high as 90%. These results clearly indicated that ni-
trate reductase CLEAs possessed good stability. The absorption at
426 nm (Ag SPR) as a function of time was measured from reaction mix-
tures of each batch cycle (Fig. S4). A comparison of time resolved absor-
bance of peak maximum at 426 nm obtained for each batch cycle shows
that the recovered nitrate reductase CLEAs exhibit almost constant cat-
alytic yield during initial time period of reaction course.
4. Conclusion
NADH-dependent nitrate reductase was directly isolated in stable
immobilized form CLEAs from crude F. oxysporum extract. It was dem-
onstrated that the type of precipitant, amount of glutaraldehyde and
cross-linking time had significant effects on the activity recovery of
enzyme in CLEAs. Immobilization improved the thermal stability of
enzyme. Based on bio-reduction ability of nitrate reductase, we demon-
strate for the first time the biocatalytic synthesis of silver nanoparticles
from silver nitrate using immobilized NADH-dependent nitrate reduc-
tase as CLEAs. Being an immobilized enzyme biocatalyst, NADH-
dependent nitrate reductase CLEAs were successfully reused for four
cycles of silver nanoparticle synthesis in batch mode.
66 S. Talekar et al. / Catalysis Communications 53 (2014) 62–66

Appendix A. Supplementary data [9] J.D. Cui, L.M. Sun, L.L. Li, Appl. Biochem. Biotechnol. 170 (2013) 1827–1837.
[10] J. Qiu, E. Su, W. Wang, D. Wei, Catal. Commun. 51 (2014) 19–23.
[11] R. Fernandez-Lafuente, Enzym. Microb. Technol. 45 (2009) 405–418.
Supplementary data to this article can be found online at http://dx. [12] D. Brady, J. Jordaan, Biotechnol. Lett. 31 (2009) 1639–1650.
doi.org/10.1016/j.catcom.2014.05.003. [13] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente,
Enzym. Microb. Technol. 40 (2007) 1451–1463.
[14] S. Talekar, A. Joshi, G. Joshi, P. Kamat, R. Haripurkar, S. Kambale, RSC Adv. 3 (2013)
References 12485–12511.
[15] J.D. Cui, S.R. Jia, Crit. Rev. Biotechnol. (2014) (in press).
[1] K.M. Koeller, C.-H. Wong, Nature 409 (2001) 232–240. [16] A.A. Makarov, I.I. Protasevich, N.P. Bazhulina, N.G. Esipova, FEBS Lett. 357 (1995) 58–61.
[2] S.A. Kumar, M.K. Abyaneh, S.W. Gosavi, S.K. Kulkarni, R. Pasricha, A. Ahmad, M.I. [17] P.V. Iyer, L. Ananthanarayan, Process Biochem. 43 (2008) 1019–1032.
Khan, Biotechnol. Lett. 29 (2007) 439–445. [18] O. Barbosa, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R.C. Rodrigues, R. Fernandez
[3] V. Deepak, K. Kalishwaralal, S. Ram Kumar Pandian, S. Gurunathan, in: M. Rai, N. Lafuente, RSC Adv. 4 (2014) 1583–1600.
Duran (Eds.), Metal nanoparticles in microbiology, Springer-Verlag, Berlin Heidel- [19] R.C. Rodrigues, Á. Berenguer-Murcia, R. Fernandez-Lafuente, Adv. Synth. Catal. 353
berg, 2011, pp. 17–35. (2011) 2216–2238.
[4] U. Roessl, J. Nahalka, B. Nidetzky, Biotechnol. Lett. 32 (2010) 341–350. [20] P. Monsan, J. Mol. Catal. 3 (1977/78) 371–384.
[5] C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-Lafuente, R.C. Rodrigues, Adv. [21] I. Matijosˇyte, I.W.C.E. Arends, S. de Vries, R.A. Sheldon, J. Mol. Catal. B Enzym. 62
Synth. Catal. 353 (2011) 2885–2904. (2010) 142–148.
[6] R.A. Sheldon, Org. Process Res. Dev. 15 (2011) 213–223. [22] R. Schoevaart, M.W. Wolbers, M. Golubovic, M.A. Ottens, P.G. Kieboom, F. Van Rantwijk,
[7] S. Talekar, V. Shah, S. Patil, M. Nimbalkar, Catal. Sci. Technol. 2 (2012) 1575–1579. L.A.M. Van der Wielen, R.A. Sheldon, Biotechnol. Bioeng. 87 (2004) 754–762.
[8] S. Talekar, S. Waingade, V. Gaikwad, S. Patil, N. Nagvekar, J. Biochem. Technol. 3 [23] C. Burda, X. Chen, R. Narayanan, M.A. EI-Sayed, Chem. Soc. Rev. 105 (2005)
(2012) 349–353. 1025–1102.