Accepted Manuscript Title: Cellulase assisted synthesis of nano silver and gold: Application as immobilization matrix for biocatalysis Author: Abhijeet Mishra Meryam Sardar PII: DOI: Reference:
S0141-8130(15)00165-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.03.014 BIOMAC 4947
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
28-11-2014 19-2-2015 9-3-2015
Please cite this article as: A. Mishra, M. Sardar, Cellulase assisted synthesis of nano silver and gold: Application as immobilization matrix for biocatalysis, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.03.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cellulase assisted synthesis of nano silver and gold: Application as immobilization matrix for biocatalysis Abhijeet Mishra and Meryam Sardar*
*Corresponding author. Phone (office): 91-11-26981717 ext: 3412,
ip t
Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India
cr
Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India
Ac ce p
te
d
M
an
us
E-mail address: [email protected]
1 Page 1 of 29
Abstract In the present study, we report in vitro synthesis of silver and gold nanoparticles (NPs) using cellulase enzyme in a single step reaction. Synthesized nanoparticles were characterized by
ip t
UV-VIS spectroscopy, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscopy (TEM), Energy-dispersive X-ray Spectroscopy (EDX), X-ray Diffraction (XRD),
cr
Circular Dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR). UV VIS studies shows absorption band at 415 nm and 520 nm for silver and gold NPs respectively
us
due to surface plasmon resonance. Sizes of NPs as shown by TEM are 5-25 nm for silver and
an
5-20 nm for gold. XRD peaks confirmed about phase purity and crystallinity of silver and gold NPs. FTIR data shows presence of amide I peak on both the NPs. The cellulase assisted
M
synthesized NPs were further exploited as immobilization matrix for cellulase enzyme. Thermal stability analysis reveals that the immobilized cellulase on synthesized NPs retained
d
77-80% activity as compared to free enzyme. While reusability data suggests immobilized
te
cellulase can be efficiently used up to sixth cycles with minimum loss of enzyme activity. The secondary structural analysis of cellulase enzyme during the synthesis of NPs and also
Ac ce p
after immobilization of cellulase on these NPs was carried out by CD spectroscopy.
Keywords: Cellulase, Biocatalysis, Nanoparticles
2 Page 2 of 29
1. Introduction In recent times, synthesis of noble metal nanoparticles (NPs) like silver and gold is gaining attention due to their unique properties and applications in various areas [1, 2]. For the
ip t
synthesis of metal NPs number of methodologies has been described in the literature [3, 4]. Broadly, they can be synthesized either by physical, chemical or biological methods [3-5].
cr
Physical and chemical methods are generally energy consuming and require toxic ingredients/hazardous materials which makes them ‘not so favoured’ methods for synthesis
us
[6, 7]. The biological method of nanoparticle synthesis is a relatively simple, cost effective
an
and environment friendly method than the conventional chemical/physical method of synthesis and thus gains an upper hand.
M
A vast array of biological resources available in nature including plants and plant products, algae, fungi, yeast, bacteria, and viruses could be employed for synthesis of NPs [2, 8, 9].
d
Biosynthetic approach has led to the fabrication of several inorganic NPs, including silver,
te
gold, copper and palladium etc. [10, 11]. Till date, the exact mechanism of nanoparticle synthesis by biological resources has not been fully understood. Studies indicate that
Ac ce p
proteins/enzymes play a major role in a biosynthesis process as they consist of number of amino acids which are reported to interact with the metal ions [12-15]. Xie et al. [16] demonstrated that proteins are the principal biomolecules which are involved in the algal synthesis of gold NPs. Ahmad et al. [17] postulated that a NADPH dependent reductase is involved in silver nanoparticle synthesis by Fusarium oxysporum. However, the biochemical mechanism of metal ion reduction and subsequent nanoparticle formation remains unexplored. Eby et al. [18] described that hen egg white lysozyme in the presence of light can act as the sole reducing and capping agent for the formation of silver NPs. Rangnekar et al. [19] described the synthesis of gold NPs using a pure enzyme alpha amylase for the reduction of AuCl4-, with the retention of enzymatic activity in the complex. They
3 Page 3 of 29
suggested that the presence of free and exposed –SH groups is essential in the reduction of AuCl4- to gold NPs. Ravindra [20] reported that lysine from serrapeptase enzyme is involved in the synthesis and stabilization of gold NPs. Gupta et al. [21] describe the synthesis of
ip t
silver NPs using native BSA and DTNB modified BSA. They found that modified and unmodified BSA are equally good for the biosynthesis of silver NPs [21]. In our lab we have
cr
synthesized and characterized gold and silver NPs using an enzyme alpha amylase from A. oryzae [3, 22]. Recently, we reported the synthesis of silver NPs using neem leaf extract
us
containing alpha amylase enzyme [23]. This enzyme activity was retained on the NPs during
an
synthesis. Several studies on immobilization of enzymes have shown that stability and reusability of enzymes increases after immobilization on nanomaterials [24, 25]. Therefore,
M
in the present study synthesis of silver and gold NPs using cellulase enzyme has been described. The boiosynthesized NPs were further exploited as immobilization matrix for
2.1 Materials
te
2. Experimental
d
cellulase enzyme.
Ac ce p
Cellulase (from A. niger) and auric chloride were procured from Sisco Research Laboratories (SRL), Mumbai, India. Silver nitrate was purchased from Merck India Ltd. Carboxy Methyl Cellulose Sodium Salt (CMC) was obtained from Qualigens Fine Chemicals Pvt. Ltd. All other chemicals and solvents used were of analytical grade and used without further purification.
2.2 Biosynthesis of silver and gold NPs by cellulase enzyme Biosynthesis of the silver and gold NPs was carried out by incubating 10 ml of cellulase enzyme (1 mg/ml for silver and 2 mg/ml for gold in Tris-HCl buffer, pH 8.0) and 90 ml of freshly prepared aqueous solution of silver nitrate (1mM)/auric chloride (1mM) in each case.
4 Page 4 of 29
Solutions were kept at 25°C and the syntheses of NPs were monitored by UV-VIS spectroscopy. The biosynthesized NPs were purified as described earlier [26]. 2.3 Characterization of purified NPs
ip t
Dynamic light scattering (DLS) measurements were done by using the Spectroscatter RiNA, GmbH class3B at 20°C for 10 cycles. Samples for transmission electron microscopy (TEM)
cr
were prepared by drop coating purified solution of silver and gold NPs on to carbon-coated copper TEM grids. TEM measurements were performed on a JEOL, F2100 instrument
us
operated at an accelerating voltage at 200kV. An EDX (Model EVO-40, ZEISS) spectrum
an
was also recorded for elemental analysis of above prepared sample. XRD patterns of both NPs were recorded by X’Pert Pro X-ray diffractometer (PANanlytical BV) by operating X-
M
ray tube at 45kV and 35mA and radiation used was Cu-Kα. Fourier transform infrared (FTIR) spectra were recorded with a Shimadzu, FTIR spectrophotometer between 4000 and 400
d
cm−1, with a resolution of 4 cm−1. Circular Dichroism (CD) measurements of free cellulase,
te
cellulase assisted silver and gold NPs and immobilized cellulase on silver and gold NPs in sodium acetate buffer (pH 4.5, 50 mM) were carried out on Applied Photophysics Circular
Ac ce p
Dichroism spectropolarimeter using 1 nm/10 sec signal. The CD instrument was consistently calibrated with D-10 camphorsulfonic acid and N2 purging was continuously done in the lamp, optics and sample chamber in a ratio of 1:3:1. Each spectrum was corrected for blank contribution.
2.4 Activity measurement of cellulase enzyme Cellulase activity in free enzyme and immobilized cellulase on silver and gold NPs were measured by using CMC as a substrate [27]. The immobilized cellulase on silver and gold NPs were continuously shaken during the assay. One unit of enzyme is defined as the amount of enzyme required to produce 1 µmol of reducing sugar per min under defined conditions. The amounts of reducing sugar were estimated using the dinitrosalicyclic acid methods [28].
5 Page 5 of 29
2.5 Protein estimation Amount of protein was estimated by the dye binding method (Bradford dye), using bovine serum albumin as the standard [29].
ip t
2.6 Immobilization of cellulase on silver and gold NPs Immobilization of cellulase on silver and gold NPs was carried out by physical adsorption
cr
method. Cellulase enzyme solutions (containing 680U for silver and 350U for gold, each dissolved in 50mM sodium acetate buffer, pH 4.5) were incubated with 5mg of silver and
us
gold NPs respectively, suspended in 1ml of 50mM sodium acetate buffer, pH 4.5. The
an
mixtures were incubated at 25°C with constant shaking after 1 h they were centrifuged at 9000 rpm for 5 min at 4°C. The silver and gold NPs containing the adsorbed enzymes were
M
washed with 50 mM sodium acetate buffer pH 4.5 containing 1M NaCl and 50% Ethylene glycol to remove the loosely bound enzyme. The enzyme activities on both the NPs were
d
determined in the supernatants and washings. To calculate and estimate the immobilization
te
efficiency on both the NPs, the enzyme loads were varied for silver (220-1200U), for gold (175-1100U) and the immobilization was carried as above. All the experiments were
Ac ce p
conducted in the batch mode.
2.7 Thermal stability of free enzyme and immobilized cellulase on silver and gold NPs Free enzyme and immobilized cellulase on silver and gold NPs were incubated in 50 mM sodium acetate buffer (pH 4.5) at 75oC for 60 min and residual activities were assayed using CMC as the substrate in each case. An appropriate aliquot of free enzyme and immobilized cellulase on silver and gold NPs (containing 680U for silver and 350U for gold) were withdrawn at various time intervals of incubation, cooled to 25°C and their activities were determined.
6 Page 6 of 29
2.8 Reusability of the immobilized cellulase on silver and gold NPs The immobilized cellulase on silver and gold NPs (680U for silver and 350U for gold) were made to 0.5 ml with the assay buffer and incubated with 0.5 ml of the substrate under shaking
ip t
condition at 50oC, separately. After 30 min the supernatants were removed by centrifugation at 9000 rpm for 5 min at 4oC and the enzyme activities were estimated in the supernatants.
cr
The immobilized cellulase on silver and gold NPs was washed three times with 1 ml of assay buffer. For second cycle the immobilized cellulase on silver and gold NPs were again
us
incubated with 0.5 ml of fresh substrate and the reaction was carried out as before in both the
an
immobilized enzymes. 3. Results and discussion
M
In the present study, noble metal NPs like silver and gold were biosynthesized using cellulase enzyme (from A. niger). Extracellular biosynthesis of silver [30] and gold NPs [31] has been
d
reported earlier also using A. niger. Similarly, synthesis of silver and gold NPs using
te
extracellular secretions of A. niger has been reported by Jaidav and Narasimha [32]. They suggested the role of different extracellular enzymes/proteins present in the supernatant
Ac ce p
catalysis the synthesis, especially the involvement of nitrate reductase has been described by them. Several other investigators also claimed that during the biosynthesis of silver and gold NPs using fungal secretions major players are the proteins/enzymes [30-32]. Cellulase from A. niger is also an extracellular enzyme, it catalyze the multistep hydrolysis of cellulose to glucose [33]. The cellulase enzymes are used in textile, food, leather, paper and pulp industries [34]. Therefore, we exploited this enzyme for the synthesis of silver and gold NPs. When the cellulase enzyme solutions were added separately with freshly prepared aqueous solutions (1mM) of silver nitrate and auric chloride, the mixture turned brown in case of silver; and ruby red in the case of gold which indicates the bioreduction of metal ions. Fig. 1a
7 Page 7 of 29
UV-VIS spectra of silver NPs at different time interval are shown in Fig 1a. The absorption centered around 415 nm due to surface plasmon resonance of silver NPs and the absorption intensity increases up to 12 h [3, 23]. However at 14 h, slight decrease in the absorption
ip t
intensity (from 1.034-0.973) was observed for silver NPs which indicate the synthesis is completed in 12 h only.
cr
Fig. 1b
UV-VIS spectra of gold NPs (Fig 1b) at different time interval shows the absorption peak at
us
520 nm which is known to arise due to surface plasmon oscillations specific to gold NPs, it also increases up to 12 h and no further increase in absorbance was observed when time was
an
increased from 12 to 14 h (Fig 1b). This increase in absorbance intensity due to surface plasmon resonance with increase in time has been reported earlier also by other researchers
M
[35, 36]. The interaction time of enzyme/reducing agents and metal ions play an important
d
role in controlling the size, shape and stability of NPs [35]. While in control experiments the
te
spectra of silver nitrate and auric chloride solutions were taken at different time interval; they did not show any peak demonstrating that cellulase enzyme is responsible for the synthesis of
Ac ce p
silver as well as gold NPs. Further, the UV-VIS spectra of only cellulase enzyme show an intense peak around 280 nm which is the characteristic peak for proteins. The absorption peak around 280 nm is also noticed during the biosynthesis of silver and gold NPs (Fig 1a and 1b) which also increases as the synthesis time are increased [3, 37]. This suggested that with increase in time more and more protein/ enzyme are bound to the NPs. Fig. 2a and 2b
DLS analyses of the cellulase assisted silver and gold NPs were performed to observe the size of NPs in solution. Fig 2a shows particle size ranges from 10-50 nm for silver NPs and 5-40 nm for gold NPs (Fig 2b). The NPs were purified by a simple process of centrifugation and the particles were washed to remove the unbound enzyme.
8 Page 8 of 29
Fig. 2c and 2d
The NPs formed were also characterized by TEM. The silver NPs formed were spherical and cubical in shape and their size ranges from 5-25 nm (Fig 2c) while gold NPs are 5-20 nm in
ip t
size and almost spherical in shape (Fig 2d). Fig. 2e and 2f
cr
The elemental compositions of the cellulase assisted silver and gold NPs were determined by EDX (Fig 2e and 2f). Elemental silver and gold signal confirms presence of metallic silver
us
and gold and no impurity was observed. The crystalline nature as well as phase purity of silver and gold NPs were investigated and established by XRD.
an
Fig. 3a and 3b
Fig 3a and 3b shows XRD pattern of cellulase assisted silver and gold NPs. XRD of purified
M
powdered silver NPs have peaks at [111], [200], [220] and [311] which are in close agreement with the standard value of joint committee for powder diffraction set (JCPDS)
d
data card no. 04-0783. Whereas, the XRD spectrum of gold NPs reveal peaks at [111], [200],
te
[220] and [311] matched with the standard joint committee for powder diffraction set
Ac ce p
(JCPDS) data card no. 05-2870.
Fig. 4
FTIR spectra of native cellulase enzyme along with cellulase assisted silver and gold NPs were shown in Fig 4. The intense peak around 1650 cm−1 (Amide I peak) [19, 38] is clearly shown in all the FTIR spectra, cellulase only, cellulase mediated silver and gold NPs. The broad absorption band appears in the range of 3000–3400 cm−1 is the confirmation of association intermolecular hydrogen bonds arising from –NH2 and –OH groups in cellulase enzymes [19, 39]. The stability of NPs in aqueous solution was checked after keeping them at 4°C for six months and then again the UV spectra were recorded. No changes in the spectra were observed in both the NPs (silver and gold). FTIR clearly indicates the presence of cellulase on both the NPs. To check whether the cellulase enzyme activity is retained in the 9 Page 9 of 29
cellulase assisted NPs, we determine the enzyme activity in both silver and gold NPs using carboxymethyl cellulose as a substrate. No enzyme activity was determined on both the NPs. The cellulase assisted NPs were further explored as immobilization matrix for cellulase
ip t
enzyme. Immobilization of enzymes on metal NPs generally enhances the stability of enzymes against harsh conditions and also improves its reusability [40]. Use of solid supports
cr
at nanoscale has the natural benefit that large quantity of catalytic molecules can be loaded on a matrix due to their high surface to volume ratio [41, 42]. Immobilization of cellulase
us
enzyme on nanomaterials has been reported by various investigators [43-45]. In the present
an
study, the cellulase was immobilized on cellulase assisted silver and gold NPs by a simple process of adsorption.
Table 1
M
Table 1 shows that as we increased the enzyme load, the immobilization efficiency increases.
d
The immobilization efficiency (B/A) is defined as the ratio of the measurable enzyme activity
te
in the nanobioconjugate (B) to the total bound activity (calculated by subtracting the unbound activity in the wash (A)). The maximum immobilization efficiency (0.9 for silver and 0.8 for
Ac ce p
gold) was achieved at a load of 680U and 350U respectively. As we further increase the enzyme load, the immobilization efficiency decreases for both the NPs. The variation of immobilization efficiency with increasing enzyme load observed here is similar to what has been reported earlier with other systems [25, 46]. Further work was carried out with the immobilized cellulase on silver and gold NPs showing the maximum immobilization efficiency.
Fig. 5
Fig 5 shows schematic representation of immobilization of cellulase on silver and gold NPs. Previous studies had also shown that when the proteins come in contact with NPs they get attached to them [45]. The attachment on NPs may be electrostatic, hydrophobic or specific
10 Page 10 of 29
chemical interaction between the protein and the nanoparticle. Jain et al [47] described the extracellular synthesis of silver NPs using Aspergillus flavus. They claimed that two proteins were involved in the synthesis and overall reaction is catalyzed in two steps. The first step
ip t
involves a 32 kDa protein which may be a reductase secreted by the fungal isolate, which causes reduction of silver ions into silver NPs. The second step involves 35 kDa proteins
cr
which bind with NPs and confer stability. In our case cellulase is performing both the functions; it is reducing the metal ion and also stabilizing the nanoparticle. It seems that
us
cellulase is not entrapping the whole nanoparticle when more molecules of fresh cellulase are
an
added to the metal NPs they get adsorbed to their surfaces. The fresh molecules of cellulase are either directly binding to the surface of NPs or they are forming a layer on enzyme
M
molecules already attached to the NPs as depicted in Fig 5. Fig. 6a and 6b
d
Fig 6a and 6b shows DLS graphs which clearly indicate that there is increase in the size of
te
cellulase assisted NPs after immobilization of cellulase enzyme on them. In case of silver NPs size increases from 50 nm to 160 nm, while for gold the size increases from 40 nm to
Ac ce p
200 nm. Increase in the size of NPs after immobilization of enzyme may be due to deposition of more molecules of cellulase on NPs. TEM studies were carried out to study the shape of immobilized cellulase on silver and gold NPs (fig 6c and 6d). TEM studies shows that the immobilized cellulase on silver NPs are mostly spherical, few oval shapes are also observed while the cellulase immobilized on gold NPs is spherical in shape (fig 6d). Fig. 6c and 6d Table 2
For efficient operation and applications, the reusability of immobilized cellulase on silver and gold NPs is considered to be of high significance value. Thus, the immobilized cellulase on silver and gold NPs was further assessed for reusability (Table 2). The reusability data shows 11 Page 11 of 29
that there is 22-27% loss in the immobilized enzyme activities after the 6th cycle. Similar results i.e. loss in enzyme activity after repeated usage has been reported by other researchers also [25, 48, 49]. This gradual loss in activity of immobilized cellulase on silver and gold
ip t
NPs may be due to various factors like protein denaturation, end product inhibition, and/or removal of one or more individual components of the cellulase complex. The immobilized
cr
cellulase on silver and gold NPs was studied for their temperature stability as compared to
Fig. 7
us
their soluble counterparts.
an
Fig 7 shows that free enzyme loses its activity at temperature of 75oC while immobilized cellulase on silver and gold NPs retained 77-80% of its activity after 60 min. To investigate
M
the structural changes in cellulase enzyme during biosynthesis of NPs and after immobilized cellulase on silver and gold NPs on these particles we have carried out circular dichroism.
d
Fig. 8
te
Fig 8a shows CD spectra of free cellulase, cellulase assisted silver NPs and cellulase
Ac ce p
immobilized on silver NPs. CD spectra show that there is loss of secondary structure of enzyme during synthesis of silver NPs [50]. The secondary structural analysis reveals that native cellulase contains 85.61% of α-helix which is completely lost during the synthesis of silver NPs and there is formation of β-sheet (44.79%). This may be the reason that no cellulase activity was determined in the silver NPs synthesized by cellulase. However FTIR data shows the presence of enzyme on them. When cellulase was again added to the NPs for the immobilization purpose no major change in the secondary structure was observed, there is only 5% decrease in alpha helical content as compared to the native cellulase. Whereas, in the case of gold NPs, the secondary structure i.e. 85 % α-helix is retained on the NPs during synthesis, still no enzyme activity was determined on the gold NPs. Involvement of amino acids from the active site of enzyme during synthesis of cellulase assisted gold NPs may be 12 Page 12 of 29
attributed for the loss of enzyme activity on the NPs. When cellulase was again added to gold NPs for immobilization its secondary structure is retained. 4. Conclusions
ip t
Here, we have shown that cellulase enzyme from a non pathogenic fungal strain A. niger can catalysis the synthesis of metal NPs (silver and gold). The NPs formed were crystalline in
cr
nature and highly stable. The synthesis of NPs using pure enzymes can give the idea about
us
the biochemical reactions involved in the biosynthetic pathways of synthesis. The synthesized NPs effectively adsorb the enzyme molecules and thus serve as the immobilization matrix.
an
The immobilized cellulase on silver and gold NPs is thermally more stable as compared to free enzyme. Thus, the cellulase assisted NPs can be exploited as immobilization matrix for
M
other enzymes also. Acknowledgements
d
The financial support provided by ICMR, Government of India to Abhijeet Mishra in the
Ac ce p
TEM studies.
te
form of SRF is greatly acknowledged. Authors are thankful to Dr. G. Saini, AIF, JNU for
References:
[1] P. Mohanpuria, K. Rana, S. Yadav, J. Nanopart. Res., 10 (2008) 507 - 517. [2] N. Durán, P. Marcato, M. Durán, A. Yadav, A. Gade, M. Rai, Appl. Microbiol. Biotechnol., 90 (2011) 1609-1624.
[3] A. Mishra, M. Sardar, Energy Environ. Focus, 3 (2014) 179-184. [4] K.N. Thakkar, S.S. Mhatre, R.Y. Parikh, Nanomed. Nanotech. Biol. Med., 6 (2010) 257262. [5] A.K. Mittal, Y. Chisti, U.C. Banerjee, Biotechnol. Adv., 31 (2013) 346-356.
13 Page 13 of 29
[6] S. Prabhu, E. Poulose, Int. Nano Lett., 2 (2012) 32. [7] A. Mishra, N.K. Kaushik, M. Sardar, D. Sahal, Colloids Surf., B., 111 (2013) 713-718.
Biotechnol., 69 (2006) 485-492.
cr
[9] R. Ahmad, N. Khatoon, M. Sardar, J. Proteins Proteomics, 4 (2013).
ip t
[8] D. Mandal, M. Bolander, D. Mukhopadhyay, G. Sarkar, P. Mukherjee, Appl. Microbiol.
us
[10] X. Li, H. Xu, Z.S. Chen, G. Chen, J. Nano Mat., 2011 (2011) 16.
[11] M. Sardar, A. Mishra, R. Ahmad, Sardar M, Mishra A, Ahmad R (2014) Biosynthesis of
an
Metal Nanoparticles and Their Applications, in: A. Tiwari, A.P.F. Turner (Eds.), Biosensors
M
Nanotechnology, Scrivener Publishing LLC, USA, 2014, pp. 239-266. [12] R. Prasad, J. Nanopart. Res., 2014 (2014) 8.
d
[13] K. Kalishwaralal, V. Deepak, S. Pandiana, M. Kottaisamy, S. BarathManiKanth, B.
te
Kartikeyan, S. Gurunathan, Colloids Surf., B., 77 (2010) 257 - 262.
Ac ce p
[14] I.W.S. Lin, C.N. Lok, C.M. Che, Chem. Sci., 5 (2014) 3144-3150. [15] R. Ahmad, M. Mohsin, T. Ahmad, M. Sardar, J. Hard. Mater., 283 (2015) 171-177. [16] J. Xie, J.Y. Lee, D.I.C. Wang, Y.P. Ting, Small, 3 (2007) 672-682. [17] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. Kumar, M. Sastry, Colloids Surf., B., 28 (2003) 313-318. [18] D.M. Eby, N.M. Schaeublin, K.E. Farrington, S.M. Hussain, G.R. Johnson, ACS Nano, 3 (2009) 984-994. [19] A. Rangnekar, T.K. Sarma, A.K. Singh, J. Deka, A. Ramesh, A. Chattopadhyay, Langmuir, 23 (2007) 5700-5706. 14 Page 14 of 29
[20] P. Ravindra, Mater. Sci. Eng. B, 163 (2009) 93-98. [21] S. Gautam, P. Dubey, M.N. Gupta, Colloids Surf., B., 102 (2013) 879-883.
ip t
[22] A. Mishra, M. Sardar, Energy Environ. Focus, 4 (2012) 143-146. [23] A. Mishra, R. Ahmad, V. Singh, M.N. Gupta, M. Sardar, J. Nanosci. Nanotechnol., 13
cr
(2013) 5028-5033.
us
[24] C. Garcia-Galan, Á. Berenguer-Murcia, R. Fernandez-Lafuente, R.C. Rodrigues, Adv. Synth. Catal., 353 (2011) 2885-2904.
an
[25] R. Ahmad, M. Sardar, Indian J. Biochem. Biophys., 51 (2014) 314-320.
M
[26] A. Mishra, M. Sardar, J. Nanoeng. Nanomanuf., 3 (2013) 217-219.
d
[27] T. Ghose, Pure Appl. Chem., 59 (1987) 257-268.
te
[28] G. Miller, Anal. Chem., 31 (1959) 426-428.
Ac ce p
[29] M.M. Bradford, Anal. Biochem., 72 (1976) 248-254. [30] A.K. Gade, P. Bonde, A.P. Ingle, P.D. Marcato, N. Duran, M.K. Rai, J. Biobased Mater. Bioenergy, 2 (2008) 243-247.
[31] N. Soni, S. Prakash, Rep. Parasitol., 2 (2012) 1-7. [32] L. Jaidev, G. Narasimha, Colloids Surf., B., 81 (2010) 430-433. [33] R.K. Sukumaran, R.R. Singhania, A. Pandey, J. Sci. Ind. Res., 64 (2005) 832. [34] J.S. Lupoi, E.A. Smith, Biotechnol. Bioeng., 108 (2011) 2835-2843. [35] A. Tripathy, A.M. Raichur, N. Chandrasekaran, T.C. Prathna, A. Mukherjee, J. Nanopart. Res. 12 (2009) 237-246. 15 Page 15 of 29
[36] S.C.G.K. Daniel, K. Nehru, M. Sivakumar, Curr. Nanosci. 8 (2012) 125-129. [37] S. Anil Kumar, K. Majid, S. Gosavi, S. Kulkarni, R. Pasricha, A. Ahmad, M. Khan, Biotechnol. Lett., 29 (2007) 439 - 445.
cr
[39] R. Sanghi, P. Verma, Bioresour. Technol., 100 (2009) 501 - 504.
ip t
[38] A. Mishra, R. Ahmad, M. Sardar, J. Nanoeng. Nanomanuf, 5 (2015) 37-42.
us
[40] R.C. Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R. Fernández-Lafuente, Chem. Soc. Rev., 42 (2013) 6290-6307.
an
[41] S. Chakraborti, T. Chatterjee, P. Joshi, A. Poddar, B. Bhattacharyya, S.P. Singh, V.
M
Gupta, P. Chakrabarti, Langmuir, 26 (2009) 3506-3513.
[42] K. Solanki, M.N. Gupta, New J. Chem., 35 (2011) 2551-2556.
te
d
[43] R.H.Y. Chang, J. Jang, K.C.W. Wu, Green Chem., 13 (2011) 2844-2850. [44] K. Khoshnevisan, A.-K. Bordbar, D. Zare, D. Davoodi, M. Noruzi, M. Barkhi, M.
Ac ce p
Tabatabaei, Biochem. Eng. J., 171 (2011) 669-673. [45] Y. Ikeda, A. Parashar, D.C. Bressler, Biotechnol. Bioprocess Eng., 19 (2014) 621-628. [46] I. Roy, M. Sardar, M.N. Gupta, Biochem. Eng. J., 16 (2003) 329-335. [47] N. Jain, A. Bhargava, S. Majumdar, J. Tarafdar, J. Panwar, Nanoscale, 3 (2011) 635641.
[48] C. Li, M. Yoshimoto, K. Fukunaga, K. Nakao, Bioresour. Technol., 98 (2007) 13661372. [49] J. Jordan, C.S. Kumar, C. Theegala, J. Mol. Catal. B: Enzym., 68 (2011) 139-146.
16 Page 16 of 29
[50] J. Lin, Z. Zhou, Z. Li, C. Zhang, X. Wang, K. Wang, G. Gao, P. Huang, D. Cui,
Ac ce p
te
d
M
an
us
cr
ip t
Nanoscale Res. Lett., 8 (2013) 1-7.
17 Page 17 of 29
Legends Captions: Fig. 1 UV-VIS spectra of (a) silver NPs (b) gold NPs. Fig. 2 Characterization of NPs (a) DLS result showing mean particle size of silver NPs (b)
ip t
DLS result showing mean particle size of gold NPs (c) TEM image of purified silver NPs (d) TEM image of purified gold NPs (e) EDX spectrum of silver NPs (f) EDX spectrum of gold
cr
NPs.
Fig. 3 XRD profile of purified and dried powder of (a) silver and (b) gold NPs synthesized
us
by cellulase enzyme.
an
Fig. 4 FTIR spectra of (a) only cellulase enzyme and (b) Cellulase assisted silver NPs (c) Cellulase assisted gold NPs.
M
Fig. 5 Scheme showing immobilization of cellulase on silver and gold NPs. Fig. 6 DLS of immobilized cellulase on (a) silver (b) gold NPs. (c) TEM image of
d
immobilized cellulase on silver nanoparticles (d) gold nanoparticles
te
Fig. 7 Thermal stability of free cellulase and immobilized cellulase on silver and gold NPs. Fig. 8 CD spectra of (a) free cellulase, cellulase assisted silver NPs and immobilized
Ac ce p
cellulase on silver NPs (b) free cellulase, cellulase assisted gold NPs and immobilized cellulase on gold NPs
18 Page 18 of 29
Table 1 Optimization of binding of cellulase on silver and gold NPs
220
170.5
325
162
404
265
612
285
680
350
611
278
1050
700
600
270
1200
1100
0.79
0.51
0.8
0.61
cr
90
0.9
0.81
0.581
0.39
0.5
0.24
an
175
Immobilization efficiency η=B/A For silver For gold
ip t
Total Units Bound Theoretical (A) For silver For gold
us
Unit Expressed Actual (B) For silver For gold
Table 2 Reusability of the immobilized cellulase on silver and gold NPs
2
M
te
1
Residual activity (%) for immobilized cellulase on silver NPs 100
d
No. of cycles
Residual activity (%) for immobilized cellulase on gold NPs 100
93
97
84
92
4
81
85
5
76
80
6
73
78
Ac ce p
3
19 Page 19 of 29
ip t us
cr Ac
ce pt
ed
M
an
Fig. 1(a)
Fig. 1(b)
20 Page 20 of 29
ip t us
cr Ac
ce pt
ed
M
an
Fig. 2(a)
Fig. 2(b)
21 Page 21 of 29
ip t cr
an
us
100 nm HV=200.0 kV Direct Mag: 15000X AIF-JNU
ce pt
ed
M
Fig. 2(c)
Ac
5 nm HV=200.0 kV Direct Mag: 200000X AIF-JNU
Fig. 2(d)
22 Page 22 of 29
cps/eV
5
4
Ag Cu
Ag
Cu
Ag
ip t
3
1
0 4
6
8
10 keV
12
18
20
cps/eV
M
7
16
an
Fig. 2(e)
14
us
2
cr
2
6
ed
5
4 Au Cu
Au
Cu
2
1
2
Ac
0
ce pt
3
4
6
Au
8
10 keV
12
14
16
18
20
Fig. 2(f)
23 Page 23 of 29
ip t cr us an
Ac
ce pt
ed
M
Fig. 3(a)
Fig. 3(b)
24 Page 24 of 29
ip t cr us an M Fig. 4
Ac
ce pt
ed
Cellulase enzyme + silver/gold ions
NP
Inactive cellulase
Again add cellulase
Immobilized cellulase NP
NP
Active cellulase NP= Nanoparticle
Fig. 5
25 Page 25 of 29
ip t cr us
Ac
ce pt
ed
M
an
Fig. 6(a)
Fig. 6(b)
26 Page 26 of 29
ip t cr us an M
Ac
ce pt
ed
Fig. 6(c)
Fig 6(d)
27 Page 27 of 29
ip t cr us an M Ac
ce pt
ed
Fig. 7
28 Page 28 of 29
ip t cr us an
Ac
ce pt
ed
M
Fig. 8(a)
Fig. 8(b)
29 Page 29 of 29
S0141-8130(15)00165-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.03.014 BIOMAC 4947
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
28-11-2014 19-2-2015 9-3-2015
Please cite this article as: A. Mishra, M. Sardar, Cellulase assisted synthesis of nano silver and gold: Application as immobilization matrix for biocatalysis, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.03.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cellulase assisted synthesis of nano silver and gold: Application as immobilization matrix for biocatalysis Abhijeet Mishra and Meryam Sardar*
*Corresponding author. Phone (office): 91-11-26981717 ext: 3412,
ip t
Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India
cr
Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India
Ac ce p
te
d
M
an
us
E-mail address: [email protected]
1 Page 1 of 29
Abstract In the present study, we report in vitro synthesis of silver and gold nanoparticles (NPs) using cellulase enzyme in a single step reaction. Synthesized nanoparticles were characterized by
ip t
UV-VIS spectroscopy, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscopy (TEM), Energy-dispersive X-ray Spectroscopy (EDX), X-ray Diffraction (XRD),
cr
Circular Dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR). UV VIS studies shows absorption band at 415 nm and 520 nm for silver and gold NPs respectively
us
due to surface plasmon resonance. Sizes of NPs as shown by TEM are 5-25 nm for silver and
an
5-20 nm for gold. XRD peaks confirmed about phase purity and crystallinity of silver and gold NPs. FTIR data shows presence of amide I peak on both the NPs. The cellulase assisted
M
synthesized NPs were further exploited as immobilization matrix for cellulase enzyme. Thermal stability analysis reveals that the immobilized cellulase on synthesized NPs retained
d
77-80% activity as compared to free enzyme. While reusability data suggests immobilized
te
cellulase can be efficiently used up to sixth cycles with minimum loss of enzyme activity. The secondary structural analysis of cellulase enzyme during the synthesis of NPs and also
Ac ce p
after immobilization of cellulase on these NPs was carried out by CD spectroscopy.
Keywords: Cellulase, Biocatalysis, Nanoparticles
2 Page 2 of 29
1. Introduction In recent times, synthesis of noble metal nanoparticles (NPs) like silver and gold is gaining attention due to their unique properties and applications in various areas [1, 2]. For the
ip t
synthesis of metal NPs number of methodologies has been described in the literature [3, 4]. Broadly, they can be synthesized either by physical, chemical or biological methods [3-5].
cr
Physical and chemical methods are generally energy consuming and require toxic ingredients/hazardous materials which makes them ‘not so favoured’ methods for synthesis
us
[6, 7]. The biological method of nanoparticle synthesis is a relatively simple, cost effective
an
and environment friendly method than the conventional chemical/physical method of synthesis and thus gains an upper hand.
M
A vast array of biological resources available in nature including plants and plant products, algae, fungi, yeast, bacteria, and viruses could be employed for synthesis of NPs [2, 8, 9].
d
Biosynthetic approach has led to the fabrication of several inorganic NPs, including silver,
te
gold, copper and palladium etc. [10, 11]. Till date, the exact mechanism of nanoparticle synthesis by biological resources has not been fully understood. Studies indicate that
Ac ce p
proteins/enzymes play a major role in a biosynthesis process as they consist of number of amino acids which are reported to interact with the metal ions [12-15]. Xie et al. [16] demonstrated that proteins are the principal biomolecules which are involved in the algal synthesis of gold NPs. Ahmad et al. [17] postulated that a NADPH dependent reductase is involved in silver nanoparticle synthesis by Fusarium oxysporum. However, the biochemical mechanism of metal ion reduction and subsequent nanoparticle formation remains unexplored. Eby et al. [18] described that hen egg white lysozyme in the presence of light can act as the sole reducing and capping agent for the formation of silver NPs. Rangnekar et al. [19] described the synthesis of gold NPs using a pure enzyme alpha amylase for the reduction of AuCl4-, with the retention of enzymatic activity in the complex. They
3 Page 3 of 29
suggested that the presence of free and exposed –SH groups is essential in the reduction of AuCl4- to gold NPs. Ravindra [20] reported that lysine from serrapeptase enzyme is involved in the synthesis and stabilization of gold NPs. Gupta et al. [21] describe the synthesis of
ip t
silver NPs using native BSA and DTNB modified BSA. They found that modified and unmodified BSA are equally good for the biosynthesis of silver NPs [21]. In our lab we have
cr
synthesized and characterized gold and silver NPs using an enzyme alpha amylase from A. oryzae [3, 22]. Recently, we reported the synthesis of silver NPs using neem leaf extract
us
containing alpha amylase enzyme [23]. This enzyme activity was retained on the NPs during
an
synthesis. Several studies on immobilization of enzymes have shown that stability and reusability of enzymes increases after immobilization on nanomaterials [24, 25]. Therefore,
M
in the present study synthesis of silver and gold NPs using cellulase enzyme has been described. The boiosynthesized NPs were further exploited as immobilization matrix for
2.1 Materials
te
2. Experimental
d
cellulase enzyme.
Ac ce p
Cellulase (from A. niger) and auric chloride were procured from Sisco Research Laboratories (SRL), Mumbai, India. Silver nitrate was purchased from Merck India Ltd. Carboxy Methyl Cellulose Sodium Salt (CMC) was obtained from Qualigens Fine Chemicals Pvt. Ltd. All other chemicals and solvents used were of analytical grade and used without further purification.
2.2 Biosynthesis of silver and gold NPs by cellulase enzyme Biosynthesis of the silver and gold NPs was carried out by incubating 10 ml of cellulase enzyme (1 mg/ml for silver and 2 mg/ml for gold in Tris-HCl buffer, pH 8.0) and 90 ml of freshly prepared aqueous solution of silver nitrate (1mM)/auric chloride (1mM) in each case.
4 Page 4 of 29
Solutions were kept at 25°C and the syntheses of NPs were monitored by UV-VIS spectroscopy. The biosynthesized NPs were purified as described earlier [26]. 2.3 Characterization of purified NPs
ip t
Dynamic light scattering (DLS) measurements were done by using the Spectroscatter RiNA, GmbH class3B at 20°C for 10 cycles. Samples for transmission electron microscopy (TEM)
cr
were prepared by drop coating purified solution of silver and gold NPs on to carbon-coated copper TEM grids. TEM measurements were performed on a JEOL, F2100 instrument
us
operated at an accelerating voltage at 200kV. An EDX (Model EVO-40, ZEISS) spectrum
an
was also recorded for elemental analysis of above prepared sample. XRD patterns of both NPs were recorded by X’Pert Pro X-ray diffractometer (PANanlytical BV) by operating X-
M
ray tube at 45kV and 35mA and radiation used was Cu-Kα. Fourier transform infrared (FTIR) spectra were recorded with a Shimadzu, FTIR spectrophotometer between 4000 and 400
d
cm−1, with a resolution of 4 cm−1. Circular Dichroism (CD) measurements of free cellulase,
te
cellulase assisted silver and gold NPs and immobilized cellulase on silver and gold NPs in sodium acetate buffer (pH 4.5, 50 mM) were carried out on Applied Photophysics Circular
Ac ce p
Dichroism spectropolarimeter using 1 nm/10 sec signal. The CD instrument was consistently calibrated with D-10 camphorsulfonic acid and N2 purging was continuously done in the lamp, optics and sample chamber in a ratio of 1:3:1. Each spectrum was corrected for blank contribution.
2.4 Activity measurement of cellulase enzyme Cellulase activity in free enzyme and immobilized cellulase on silver and gold NPs were measured by using CMC as a substrate [27]. The immobilized cellulase on silver and gold NPs were continuously shaken during the assay. One unit of enzyme is defined as the amount of enzyme required to produce 1 µmol of reducing sugar per min under defined conditions. The amounts of reducing sugar were estimated using the dinitrosalicyclic acid methods [28].
5 Page 5 of 29
2.5 Protein estimation Amount of protein was estimated by the dye binding method (Bradford dye), using bovine serum albumin as the standard [29].
ip t
2.6 Immobilization of cellulase on silver and gold NPs Immobilization of cellulase on silver and gold NPs was carried out by physical adsorption
cr
method. Cellulase enzyme solutions (containing 680U for silver and 350U for gold, each dissolved in 50mM sodium acetate buffer, pH 4.5) were incubated with 5mg of silver and
us
gold NPs respectively, suspended in 1ml of 50mM sodium acetate buffer, pH 4.5. The
an
mixtures were incubated at 25°C with constant shaking after 1 h they were centrifuged at 9000 rpm for 5 min at 4°C. The silver and gold NPs containing the adsorbed enzymes were
M
washed with 50 mM sodium acetate buffer pH 4.5 containing 1M NaCl and 50% Ethylene glycol to remove the loosely bound enzyme. The enzyme activities on both the NPs were
d
determined in the supernatants and washings. To calculate and estimate the immobilization
te
efficiency on both the NPs, the enzyme loads were varied for silver (220-1200U), for gold (175-1100U) and the immobilization was carried as above. All the experiments were
Ac ce p
conducted in the batch mode.
2.7 Thermal stability of free enzyme and immobilized cellulase on silver and gold NPs Free enzyme and immobilized cellulase on silver and gold NPs were incubated in 50 mM sodium acetate buffer (pH 4.5) at 75oC for 60 min and residual activities were assayed using CMC as the substrate in each case. An appropriate aliquot of free enzyme and immobilized cellulase on silver and gold NPs (containing 680U for silver and 350U for gold) were withdrawn at various time intervals of incubation, cooled to 25°C and their activities were determined.
6 Page 6 of 29
2.8 Reusability of the immobilized cellulase on silver and gold NPs The immobilized cellulase on silver and gold NPs (680U for silver and 350U for gold) were made to 0.5 ml with the assay buffer and incubated with 0.5 ml of the substrate under shaking
ip t
condition at 50oC, separately. After 30 min the supernatants were removed by centrifugation at 9000 rpm for 5 min at 4oC and the enzyme activities were estimated in the supernatants.
cr
The immobilized cellulase on silver and gold NPs was washed three times with 1 ml of assay buffer. For second cycle the immobilized cellulase on silver and gold NPs were again
us
incubated with 0.5 ml of fresh substrate and the reaction was carried out as before in both the
an
immobilized enzymes. 3. Results and discussion
M
In the present study, noble metal NPs like silver and gold were biosynthesized using cellulase enzyme (from A. niger). Extracellular biosynthesis of silver [30] and gold NPs [31] has been
d
reported earlier also using A. niger. Similarly, synthesis of silver and gold NPs using
te
extracellular secretions of A. niger has been reported by Jaidav and Narasimha [32]. They suggested the role of different extracellular enzymes/proteins present in the supernatant
Ac ce p
catalysis the synthesis, especially the involvement of nitrate reductase has been described by them. Several other investigators also claimed that during the biosynthesis of silver and gold NPs using fungal secretions major players are the proteins/enzymes [30-32]. Cellulase from A. niger is also an extracellular enzyme, it catalyze the multistep hydrolysis of cellulose to glucose [33]. The cellulase enzymes are used in textile, food, leather, paper and pulp industries [34]. Therefore, we exploited this enzyme for the synthesis of silver and gold NPs. When the cellulase enzyme solutions were added separately with freshly prepared aqueous solutions (1mM) of silver nitrate and auric chloride, the mixture turned brown in case of silver; and ruby red in the case of gold which indicates the bioreduction of metal ions. Fig. 1a
7 Page 7 of 29
UV-VIS spectra of silver NPs at different time interval are shown in Fig 1a. The absorption centered around 415 nm due to surface plasmon resonance of silver NPs and the absorption intensity increases up to 12 h [3, 23]. However at 14 h, slight decrease in the absorption
ip t
intensity (from 1.034-0.973) was observed for silver NPs which indicate the synthesis is completed in 12 h only.
cr
Fig. 1b
UV-VIS spectra of gold NPs (Fig 1b) at different time interval shows the absorption peak at
us
520 nm which is known to arise due to surface plasmon oscillations specific to gold NPs, it also increases up to 12 h and no further increase in absorbance was observed when time was
an
increased from 12 to 14 h (Fig 1b). This increase in absorbance intensity due to surface plasmon resonance with increase in time has been reported earlier also by other researchers
M
[35, 36]. The interaction time of enzyme/reducing agents and metal ions play an important
d
role in controlling the size, shape and stability of NPs [35]. While in control experiments the
te
spectra of silver nitrate and auric chloride solutions were taken at different time interval; they did not show any peak demonstrating that cellulase enzyme is responsible for the synthesis of
Ac ce p
silver as well as gold NPs. Further, the UV-VIS spectra of only cellulase enzyme show an intense peak around 280 nm which is the characteristic peak for proteins. The absorption peak around 280 nm is also noticed during the biosynthesis of silver and gold NPs (Fig 1a and 1b) which also increases as the synthesis time are increased [3, 37]. This suggested that with increase in time more and more protein/ enzyme are bound to the NPs. Fig. 2a and 2b
DLS analyses of the cellulase assisted silver and gold NPs were performed to observe the size of NPs in solution. Fig 2a shows particle size ranges from 10-50 nm for silver NPs and 5-40 nm for gold NPs (Fig 2b). The NPs were purified by a simple process of centrifugation and the particles were washed to remove the unbound enzyme.
8 Page 8 of 29
Fig. 2c and 2d
The NPs formed were also characterized by TEM. The silver NPs formed were spherical and cubical in shape and their size ranges from 5-25 nm (Fig 2c) while gold NPs are 5-20 nm in
ip t
size and almost spherical in shape (Fig 2d). Fig. 2e and 2f
cr
The elemental compositions of the cellulase assisted silver and gold NPs were determined by EDX (Fig 2e and 2f). Elemental silver and gold signal confirms presence of metallic silver
us
and gold and no impurity was observed. The crystalline nature as well as phase purity of silver and gold NPs were investigated and established by XRD.
an
Fig. 3a and 3b
Fig 3a and 3b shows XRD pattern of cellulase assisted silver and gold NPs. XRD of purified
M
powdered silver NPs have peaks at [111], [200], [220] and [311] which are in close agreement with the standard value of joint committee for powder diffraction set (JCPDS)
d
data card no. 04-0783. Whereas, the XRD spectrum of gold NPs reveal peaks at [111], [200],
te
[220] and [311] matched with the standard joint committee for powder diffraction set
Ac ce p
(JCPDS) data card no. 05-2870.
Fig. 4
FTIR spectra of native cellulase enzyme along with cellulase assisted silver and gold NPs were shown in Fig 4. The intense peak around 1650 cm−1 (Amide I peak) [19, 38] is clearly shown in all the FTIR spectra, cellulase only, cellulase mediated silver and gold NPs. The broad absorption band appears in the range of 3000–3400 cm−1 is the confirmation of association intermolecular hydrogen bonds arising from –NH2 and –OH groups in cellulase enzymes [19, 39]. The stability of NPs in aqueous solution was checked after keeping them at 4°C for six months and then again the UV spectra were recorded. No changes in the spectra were observed in both the NPs (silver and gold). FTIR clearly indicates the presence of cellulase on both the NPs. To check whether the cellulase enzyme activity is retained in the 9 Page 9 of 29
cellulase assisted NPs, we determine the enzyme activity in both silver and gold NPs using carboxymethyl cellulose as a substrate. No enzyme activity was determined on both the NPs. The cellulase assisted NPs were further explored as immobilization matrix for cellulase
ip t
enzyme. Immobilization of enzymes on metal NPs generally enhances the stability of enzymes against harsh conditions and also improves its reusability [40]. Use of solid supports
cr
at nanoscale has the natural benefit that large quantity of catalytic molecules can be loaded on a matrix due to their high surface to volume ratio [41, 42]. Immobilization of cellulase
us
enzyme on nanomaterials has been reported by various investigators [43-45]. In the present
an
study, the cellulase was immobilized on cellulase assisted silver and gold NPs by a simple process of adsorption.
Table 1
M
Table 1 shows that as we increased the enzyme load, the immobilization efficiency increases.
d
The immobilization efficiency (B/A) is defined as the ratio of the measurable enzyme activity
te
in the nanobioconjugate (B) to the total bound activity (calculated by subtracting the unbound activity in the wash (A)). The maximum immobilization efficiency (0.9 for silver and 0.8 for
Ac ce p
gold) was achieved at a load of 680U and 350U respectively. As we further increase the enzyme load, the immobilization efficiency decreases for both the NPs. The variation of immobilization efficiency with increasing enzyme load observed here is similar to what has been reported earlier with other systems [25, 46]. Further work was carried out with the immobilized cellulase on silver and gold NPs showing the maximum immobilization efficiency.
Fig. 5
Fig 5 shows schematic representation of immobilization of cellulase on silver and gold NPs. Previous studies had also shown that when the proteins come in contact with NPs they get attached to them [45]. The attachment on NPs may be electrostatic, hydrophobic or specific
10 Page 10 of 29
chemical interaction between the protein and the nanoparticle. Jain et al [47] described the extracellular synthesis of silver NPs using Aspergillus flavus. They claimed that two proteins were involved in the synthesis and overall reaction is catalyzed in two steps. The first step
ip t
involves a 32 kDa protein which may be a reductase secreted by the fungal isolate, which causes reduction of silver ions into silver NPs. The second step involves 35 kDa proteins
cr
which bind with NPs and confer stability. In our case cellulase is performing both the functions; it is reducing the metal ion and also stabilizing the nanoparticle. It seems that
us
cellulase is not entrapping the whole nanoparticle when more molecules of fresh cellulase are
an
added to the metal NPs they get adsorbed to their surfaces. The fresh molecules of cellulase are either directly binding to the surface of NPs or they are forming a layer on enzyme
M
molecules already attached to the NPs as depicted in Fig 5. Fig. 6a and 6b
d
Fig 6a and 6b shows DLS graphs which clearly indicate that there is increase in the size of
te
cellulase assisted NPs after immobilization of cellulase enzyme on them. In case of silver NPs size increases from 50 nm to 160 nm, while for gold the size increases from 40 nm to
Ac ce p
200 nm. Increase in the size of NPs after immobilization of enzyme may be due to deposition of more molecules of cellulase on NPs. TEM studies were carried out to study the shape of immobilized cellulase on silver and gold NPs (fig 6c and 6d). TEM studies shows that the immobilized cellulase on silver NPs are mostly spherical, few oval shapes are also observed while the cellulase immobilized on gold NPs is spherical in shape (fig 6d). Fig. 6c and 6d Table 2
For efficient operation and applications, the reusability of immobilized cellulase on silver and gold NPs is considered to be of high significance value. Thus, the immobilized cellulase on silver and gold NPs was further assessed for reusability (Table 2). The reusability data shows 11 Page 11 of 29
that there is 22-27% loss in the immobilized enzyme activities after the 6th cycle. Similar results i.e. loss in enzyme activity after repeated usage has been reported by other researchers also [25, 48, 49]. This gradual loss in activity of immobilized cellulase on silver and gold
ip t
NPs may be due to various factors like protein denaturation, end product inhibition, and/or removal of one or more individual components of the cellulase complex. The immobilized
cr
cellulase on silver and gold NPs was studied for their temperature stability as compared to
Fig. 7
us
their soluble counterparts.
an
Fig 7 shows that free enzyme loses its activity at temperature of 75oC while immobilized cellulase on silver and gold NPs retained 77-80% of its activity after 60 min. To investigate
M
the structural changes in cellulase enzyme during biosynthesis of NPs and after immobilized cellulase on silver and gold NPs on these particles we have carried out circular dichroism.
d
Fig. 8
te
Fig 8a shows CD spectra of free cellulase, cellulase assisted silver NPs and cellulase
Ac ce p
immobilized on silver NPs. CD spectra show that there is loss of secondary structure of enzyme during synthesis of silver NPs [50]. The secondary structural analysis reveals that native cellulase contains 85.61% of α-helix which is completely lost during the synthesis of silver NPs and there is formation of β-sheet (44.79%). This may be the reason that no cellulase activity was determined in the silver NPs synthesized by cellulase. However FTIR data shows the presence of enzyme on them. When cellulase was again added to the NPs for the immobilization purpose no major change in the secondary structure was observed, there is only 5% decrease in alpha helical content as compared to the native cellulase. Whereas, in the case of gold NPs, the secondary structure i.e. 85 % α-helix is retained on the NPs during synthesis, still no enzyme activity was determined on the gold NPs. Involvement of amino acids from the active site of enzyme during synthesis of cellulase assisted gold NPs may be 12 Page 12 of 29
attributed for the loss of enzyme activity on the NPs. When cellulase was again added to gold NPs for immobilization its secondary structure is retained. 4. Conclusions
ip t
Here, we have shown that cellulase enzyme from a non pathogenic fungal strain A. niger can catalysis the synthesis of metal NPs (silver and gold). The NPs formed were crystalline in
cr
nature and highly stable. The synthesis of NPs using pure enzymes can give the idea about
us
the biochemical reactions involved in the biosynthetic pathways of synthesis. The synthesized NPs effectively adsorb the enzyme molecules and thus serve as the immobilization matrix.
an
The immobilized cellulase on silver and gold NPs is thermally more stable as compared to free enzyme. Thus, the cellulase assisted NPs can be exploited as immobilization matrix for
M
other enzymes also. Acknowledgements
d
The financial support provided by ICMR, Government of India to Abhijeet Mishra in the
Ac ce p
TEM studies.
te
form of SRF is greatly acknowledged. Authors are thankful to Dr. G. Saini, AIF, JNU for
References:
[1] P. Mohanpuria, K. Rana, S. Yadav, J. Nanopart. Res., 10 (2008) 507 - 517. [2] N. Durán, P. Marcato, M. Durán, A. Yadav, A. Gade, M. Rai, Appl. Microbiol. Biotechnol., 90 (2011) 1609-1624.
[3] A. Mishra, M. Sardar, Energy Environ. Focus, 3 (2014) 179-184. [4] K.N. Thakkar, S.S. Mhatre, R.Y. Parikh, Nanomed. Nanotech. Biol. Med., 6 (2010) 257262. [5] A.K. Mittal, Y. Chisti, U.C. Banerjee, Biotechnol. Adv., 31 (2013) 346-356.
13 Page 13 of 29
[6] S. Prabhu, E. Poulose, Int. Nano Lett., 2 (2012) 32. [7] A. Mishra, N.K. Kaushik, M. Sardar, D. Sahal, Colloids Surf., B., 111 (2013) 713-718.
Biotechnol., 69 (2006) 485-492.
cr
[9] R. Ahmad, N. Khatoon, M. Sardar, J. Proteins Proteomics, 4 (2013).
ip t
[8] D. Mandal, M. Bolander, D. Mukhopadhyay, G. Sarkar, P. Mukherjee, Appl. Microbiol.
us
[10] X. Li, H. Xu, Z.S. Chen, G. Chen, J. Nano Mat., 2011 (2011) 16.
[11] M. Sardar, A. Mishra, R. Ahmad, Sardar M, Mishra A, Ahmad R (2014) Biosynthesis of
an
Metal Nanoparticles and Their Applications, in: A. Tiwari, A.P.F. Turner (Eds.), Biosensors
M
Nanotechnology, Scrivener Publishing LLC, USA, 2014, pp. 239-266. [12] R. Prasad, J. Nanopart. Res., 2014 (2014) 8.
d
[13] K. Kalishwaralal, V. Deepak, S. Pandiana, M. Kottaisamy, S. BarathManiKanth, B.
te
Kartikeyan, S. Gurunathan, Colloids Surf., B., 77 (2010) 257 - 262.
Ac ce p
[14] I.W.S. Lin, C.N. Lok, C.M. Che, Chem. Sci., 5 (2014) 3144-3150. [15] R. Ahmad, M. Mohsin, T. Ahmad, M. Sardar, J. Hard. Mater., 283 (2015) 171-177. [16] J. Xie, J.Y. Lee, D.I.C. Wang, Y.P. Ting, Small, 3 (2007) 672-682. [17] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. Kumar, M. Sastry, Colloids Surf., B., 28 (2003) 313-318. [18] D.M. Eby, N.M. Schaeublin, K.E. Farrington, S.M. Hussain, G.R. Johnson, ACS Nano, 3 (2009) 984-994. [19] A. Rangnekar, T.K. Sarma, A.K. Singh, J. Deka, A. Ramesh, A. Chattopadhyay, Langmuir, 23 (2007) 5700-5706. 14 Page 14 of 29
[20] P. Ravindra, Mater. Sci. Eng. B, 163 (2009) 93-98. [21] S. Gautam, P. Dubey, M.N. Gupta, Colloids Surf., B., 102 (2013) 879-883.
ip t
[22] A. Mishra, M. Sardar, Energy Environ. Focus, 4 (2012) 143-146. [23] A. Mishra, R. Ahmad, V. Singh, M.N. Gupta, M. Sardar, J. Nanosci. Nanotechnol., 13
cr
(2013) 5028-5033.
us
[24] C. Garcia-Galan, Á. Berenguer-Murcia, R. Fernandez-Lafuente, R.C. Rodrigues, Adv. Synth. Catal., 353 (2011) 2885-2904.
an
[25] R. Ahmad, M. Sardar, Indian J. Biochem. Biophys., 51 (2014) 314-320.
M
[26] A. Mishra, M. Sardar, J. Nanoeng. Nanomanuf., 3 (2013) 217-219.
d
[27] T. Ghose, Pure Appl. Chem., 59 (1987) 257-268.
te
[28] G. Miller, Anal. Chem., 31 (1959) 426-428.
Ac ce p
[29] M.M. Bradford, Anal. Biochem., 72 (1976) 248-254. [30] A.K. Gade, P. Bonde, A.P. Ingle, P.D. Marcato, N. Duran, M.K. Rai, J. Biobased Mater. Bioenergy, 2 (2008) 243-247.
[31] N. Soni, S. Prakash, Rep. Parasitol., 2 (2012) 1-7. [32] L. Jaidev, G. Narasimha, Colloids Surf., B., 81 (2010) 430-433. [33] R.K. Sukumaran, R.R. Singhania, A. Pandey, J. Sci. Ind. Res., 64 (2005) 832. [34] J.S. Lupoi, E.A. Smith, Biotechnol. Bioeng., 108 (2011) 2835-2843. [35] A. Tripathy, A.M. Raichur, N. Chandrasekaran, T.C. Prathna, A. Mukherjee, J. Nanopart. Res. 12 (2009) 237-246. 15 Page 15 of 29
[36] S.C.G.K. Daniel, K. Nehru, M. Sivakumar, Curr. Nanosci. 8 (2012) 125-129. [37] S. Anil Kumar, K. Majid, S. Gosavi, S. Kulkarni, R. Pasricha, A. Ahmad, M. Khan, Biotechnol. Lett., 29 (2007) 439 - 445.
cr
[39] R. Sanghi, P. Verma, Bioresour. Technol., 100 (2009) 501 - 504.
ip t
[38] A. Mishra, R. Ahmad, M. Sardar, J. Nanoeng. Nanomanuf, 5 (2015) 37-42.
us
[40] R.C. Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R. Fernández-Lafuente, Chem. Soc. Rev., 42 (2013) 6290-6307.
an
[41] S. Chakraborti, T. Chatterjee, P. Joshi, A. Poddar, B. Bhattacharyya, S.P. Singh, V.
M
Gupta, P. Chakrabarti, Langmuir, 26 (2009) 3506-3513.
[42] K. Solanki, M.N. Gupta, New J. Chem., 35 (2011) 2551-2556.
te
d
[43] R.H.Y. Chang, J. Jang, K.C.W. Wu, Green Chem., 13 (2011) 2844-2850. [44] K. Khoshnevisan, A.-K. Bordbar, D. Zare, D. Davoodi, M. Noruzi, M. Barkhi, M.
Ac ce p
Tabatabaei, Biochem. Eng. J., 171 (2011) 669-673. [45] Y. Ikeda, A. Parashar, D.C. Bressler, Biotechnol. Bioprocess Eng., 19 (2014) 621-628. [46] I. Roy, M. Sardar, M.N. Gupta, Biochem. Eng. J., 16 (2003) 329-335. [47] N. Jain, A. Bhargava, S. Majumdar, J. Tarafdar, J. Panwar, Nanoscale, 3 (2011) 635641.
[48] C. Li, M. Yoshimoto, K. Fukunaga, K. Nakao, Bioresour. Technol., 98 (2007) 13661372. [49] J. Jordan, C.S. Kumar, C. Theegala, J. Mol. Catal. B: Enzym., 68 (2011) 139-146.
16 Page 16 of 29
[50] J. Lin, Z. Zhou, Z. Li, C. Zhang, X. Wang, K. Wang, G. Gao, P. Huang, D. Cui,
Ac ce p
te
d
M
an
us
cr
ip t
Nanoscale Res. Lett., 8 (2013) 1-7.
17 Page 17 of 29
Legends Captions: Fig. 1 UV-VIS spectra of (a) silver NPs (b) gold NPs. Fig. 2 Characterization of NPs (a) DLS result showing mean particle size of silver NPs (b)
ip t
DLS result showing mean particle size of gold NPs (c) TEM image of purified silver NPs (d) TEM image of purified gold NPs (e) EDX spectrum of silver NPs (f) EDX spectrum of gold
cr
NPs.
Fig. 3 XRD profile of purified and dried powder of (a) silver and (b) gold NPs synthesized
us
by cellulase enzyme.
an
Fig. 4 FTIR spectra of (a) only cellulase enzyme and (b) Cellulase assisted silver NPs (c) Cellulase assisted gold NPs.
M
Fig. 5 Scheme showing immobilization of cellulase on silver and gold NPs. Fig. 6 DLS of immobilized cellulase on (a) silver (b) gold NPs. (c) TEM image of
d
immobilized cellulase on silver nanoparticles (d) gold nanoparticles
te
Fig. 7 Thermal stability of free cellulase and immobilized cellulase on silver and gold NPs. Fig. 8 CD spectra of (a) free cellulase, cellulase assisted silver NPs and immobilized
Ac ce p
cellulase on silver NPs (b) free cellulase, cellulase assisted gold NPs and immobilized cellulase on gold NPs
18 Page 18 of 29
Table 1 Optimization of binding of cellulase on silver and gold NPs
220
170.5
325
162
404
265
612
285
680
350
611
278
1050
700
600
270
1200
1100
0.79
0.51
0.8
0.61
cr
90
0.9
0.81
0.581
0.39
0.5
0.24
an
175
Immobilization efficiency η=B/A For silver For gold
ip t
Total Units Bound Theoretical (A) For silver For gold
us
Unit Expressed Actual (B) For silver For gold
Table 2 Reusability of the immobilized cellulase on silver and gold NPs
2
M
te
1
Residual activity (%) for immobilized cellulase on silver NPs 100
d
No. of cycles
Residual activity (%) for immobilized cellulase on gold NPs 100
93
97
84
92
4
81
85
5
76
80
6
73
78
Ac ce p
3
19 Page 19 of 29
ip t us
cr Ac
ce pt
ed
M
an
Fig. 1(a)
Fig. 1(b)
20 Page 20 of 29
ip t us
cr Ac
ce pt
ed
M
an
Fig. 2(a)
Fig. 2(b)
21 Page 21 of 29
ip t cr
an
us
100 nm HV=200.0 kV Direct Mag: 15000X AIF-JNU
ce pt
ed
M
Fig. 2(c)
Ac
5 nm HV=200.0 kV Direct Mag: 200000X AIF-JNU
Fig. 2(d)
22 Page 22 of 29
cps/eV
5
4
Ag Cu
Ag
Cu
Ag
ip t
3
1
0 4
6
8
10 keV
12
18
20
cps/eV
M
7
16
an
Fig. 2(e)
14
us
2
cr
2
6
ed
5
4 Au Cu
Au
Cu
2
1
2
Ac
0
ce pt
3
4
6
Au
8
10 keV
12
14
16
18
20
Fig. 2(f)
23 Page 23 of 29
ip t cr us an
Ac
ce pt
ed
M
Fig. 3(a)
Fig. 3(b)
24 Page 24 of 29
ip t cr us an M Fig. 4
Ac
ce pt
ed
Cellulase enzyme + silver/gold ions
NP
Inactive cellulase
Again add cellulase
Immobilized cellulase NP
NP
Active cellulase NP= Nanoparticle
Fig. 5
25 Page 25 of 29
ip t cr us
Ac
ce pt
ed
M
an
Fig. 6(a)
Fig. 6(b)
26 Page 26 of 29
ip t cr us an M
Ac
ce pt
ed
Fig. 6(c)
Fig 6(d)
27 Page 27 of 29
ip t cr us an M Ac
ce pt
ed
Fig. 7
28 Page 28 of 29
ip t cr us an
Ac
ce pt
ed
M
Fig. 8(a)
Fig. 8(b)
29 Page 29 of 29
