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Effect of changing the nanoscale environment on activity and stability of nitrate reductase
Accepted Manuscript Title: Effect of changing the nanoscale environment on activity and stability of nitrate reductase Author: Veena Sachdeva Vinita Hooda PII: DOI: Reference:
S0141-0229(16)30052-7 http://dx.doi.org/doi:10.1016/j.enzmictec.2016.03.007 EMT 8889
To appear in:
Enzyme and Microbial Technology
Received date: Revised date: Accepted date:
23-9-2015 6-3-2016 21-3-2016
Please cite this article as: Sachdeva Veena, Hooda Vinita.Effect of changing the nanoscale environment on activity and stability of nitrate reductase.Enzyme and Microbial Technology http://dx.doi.org/10.1016/j.enzmictec.2016.03.007 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.
Effect of changing the nanoscale environment on activity and stability of nitrate reductase
Veena Sachdeva, Vinita Hooda* 1
Department of Botany, Faculty of Life Sciences, Maharshi Dayanand University Rohtak-124001, India
*Corresponding author. Tel: +91 9896795000; fax: +91 1262 247150 Email: [email protected] 1
Postal address
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Highlights
Nitrate reductase (NR) covalently immobilized onto epoxy affixed Fe3O4 and ZnO nanoparticles.
High immobilization efficiency with decreased Km values was achieved.
Storage and thermal stabilities improved substantially after immobilization.
Nitrate determination using immobilized NR produced reliable data.
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Abstract Nitrate reductase (NR) is employed for fabrication of nitrate sensing devices in which the enzyme in immobilized form is used to catalyze the conversion of nitrate to nitrite in the presence of a suitable cofactor. So far, instability of immobilized NR due to the use of inappropriate immobilization matrices has limited the practical applications of these devices. Present study is an attempt to improve the kinetic properties and stability of NR using nanoscale iron oxide (nFe3O4) and zinc oxide (nZnO) particles. The desired nanoparticles were synthesized, surface functionalized, characterized and affixed onto the epoxy resin to yield two nanocomposite supports (epoxy/nFe3O4 and epoxy/nZnO) for immobilizing NR. Epoxy/nFe3O4 and epoxy/nZnO support could load as much as 35.8±0.01 and 33.20±0.01 μg/cm2 of NR with retention of about 93.72±0.50 and 84.81±0.80 % of its initial activity respectively. Changes in surface morphology and chemical bonding structure of both the nanocomposite supports after addition of NR were confirmed by scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR). Optimum working conditions of pH, temperature and substrate concentration were ascertained for free as well as immobilized NR preparations. Further, storage stability at 4 °C and thermal stability between 25 to 50 °C were determined for all the NR preparations. Analytical applications of immobilized NR for determination of soil and water nitrates along with reusability data has been included to make sure the usefulness of the procedure.
Keywords: Nitrate reductase; Zinc oxide; Iron oxide; Nanoparticles; Immobilization; Nitrate determination
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1. Introduction Nitrate reductase (NR) is a complex multiredox center enzyme that catalyzes the reduction of nitrate to nitrite in the presence of NADH or other suitable cofactor/mediator as shown in equation (i): NO3 + NAD(P)H + H+
NO2 + NAD(P)+ + ½ H2O
----------(i)
The enzyme (NR) is usually immobilized onto conducting polymers like polypyrrole [13], alkylpyrroleviologen [4], polyviologen [5,6], methyl viologen and azure A [7-9] for construction of nitrate biosensor. Since, the redox centers of NR are deeply embedded in the protein structure, conducting polymers help to achieve better electrical wiring of enzyme to the electrode surface. On the downside, partial hydrophobic character of these immobilization matrices impose conformational constraints in the protein structure and exert diffusional restrictions within the microenvironment of bound enzyme, which greatly affects not only the activity, but also the stability of the bound enzyme [10]. Moreover, in majority of the studies, NR is weakly bonded to these matrices as immobilization is carried out by adsorption and/or entrapment. As a result, most of the nitrate biosensors perform poorly either due to denaturation of enzyme or leaching of immobilized enzyme from the polymers. Sometimes, strong bonding of NR with polymer film decreases the number of free active sites, effectively decreasing the rate of catalytic reaction [11]. Hence, the importance of a suitable immobilization matrix for stabilizing NR can no longer be ignored. Over the past few years, great interest has been generated in the use of metal and metal oxide nanoparticles for enzyme stabilization [12]. Although both metal and metal oxide nanoparticles have yielded promising results but metal oxide nanoparticles have the advantage of being more reactive due to the presence of uncoordinated atoms on their corners and edges. Earlier NR has been immobilized onto silver [13] and gold [14]
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nanoparticles with considerable improvement in its properties, but so far it has not been immobilized onto any metal oxide nanoparticle. For this purpose, ZnO and Fe3O4 nanoparticles (nZnO and nFe3O4) are of special interest. Since nZnO has the unique ability to display different geometric nanostructures with large interfacial areas, thereby scaling up the amount of loaded enzyme and paramagnetic nature of nFe3O4 offers ease of separation under external magnetic field [15]. In addition, their biocompatible nature ensures that the loaded enzyme remains biologically active and good electrical conductivity helps in faster electron transfer to and from the active centres of enzymes. Both nZnO and nFe3O4 have so far been utilized for immobilization of a number of enzymes including glucose oxidase [16,17], cholesterol oxidase [18,19], lipase [20,21], β galactosidase [22,23] and urease [24,25] with increased activity, stability, reuse capacity and storage stability. Usually, these immobilization protocols rely on separation of nanoparticles from the solution either by centrifugation in case of nZnO or by applying external magnetic field for nFe3O4. Alternatively, the nanoparticles can be affixed onto some stable insoluble matrix such as epoxy, which will not only make the isolation of nanoparticles from solution easy but will also impart structural stability to the matrix [14]. Hence, in the present study, suitability of epoxy affixed ZnO and Fe3O4 nanoparticles (epoxy/nZnO and epoxy/nFe3O4 respectively) as immobilization supports for NR has been studied. Changes in optimum pH, optimum temperature, kinetics and stability of NR after immobilization vis a free NR are reported. Finally, analytical applications of both the immobilized NR preparations are demonstrated.
2. Materials and methods 2.1. Chemicals
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11-mercaptoundecanoic acid (MUA), nitrate reductase (NR; NAD(P)H) from Aspergillus niger (≥ 300.0 units/g), N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), β-nicotinamide adenine dinucleotide (ß-NADH), bovine serum albumin (BSA), FeCl3·6H2O and FeCl2.4H2O were purchased from Sigma-Aldrich Co. (St. Louis, USA). N(1-naphthyl)
ethylenediaminedihydrochloride
(NEDA),
tri-sodium
citrate,
N-
hydroxysuccinimide (NHS), sulfanilamide and tween 20 were obtained from Himedia (Mumbai, India). Ethylenediaminetetraacetic acid (EDTA) and zinc nitrate were procured from Thomas Baker (Mumbai, India). Epoxy resin and bisphenol A available as a popular adhesive under the trade name “Araldite” were purchased from the local market and epoxy layer was prepared by mixing both the components in 1:1 ratio, as per the directions written on pack. All other chemicals purchased were of analytical reagent (AR) grade. Deionized water was used as solvent in all the experiments.
2.2. Synthesis of nFe3O4
Iron oxide nanoparticles were prepared by co-precipitating di and trivalent Fe ions by alkaline solution as described by Khatiri et al. (2013) [26]. FeCl3·6H2O (2.70 g) and FeCl2·4H2O (1.00 g) were dissolved into 50 mL of water. To this solution, 25 mL of 30% ammonia was added at 80 ⁰C under nonmagnetic stirring. The stirring was continued for 30 min and the reaction mixture was cooled to room temperature. The precipitate was separated by washing and centrifuging several times in water and then in ethanol at 2800 rpm. The nanoparticles were finally dried in air.
2.3. Synthesis of nZnO
To prepare nZnO, 0.45 M aqueous solution of zinc nitrate and 0.9 M aqueous solution of NaOH were prepared. The beaker containing NaOH solution was heated upto 55 °C and zinc
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nitrate solution was added drop wise for 40 min to the heated solution under high speed stirring. The beaker was sealed at this condition for 2 h. The precipitated nZnO were cleaned with deionized water and ethanol and air-dried at 60 °C [27].
2.4. Characterization of nanoparticles Size of nanoparticles was confirmed by Transmission Electron Microscopy (TEM – Jeol 2100F) at Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi and the crystal structure was illucidated by XRD (Panalytical's X'Pert Pro) at Sophisticated Analytical Instrumentation Facility, Punjab University Chandigarh.
2.5. Surface modification and characterization of nanoparticles In order to prevent the aggregation of nanoparticles and improve their solubility, the surface of nanoparticles was first modified with MUA, a functional thiol and then MUA terminated nanoparticles were cross-linked to amino groups on the surface of enzyme using EDC/NHS coupling reaction. To prepare surface modified nanoparticles, a suspension of 5.0 mg of nanoparticles in 5.0 ml of phosphate buffer (10.0 mM, pH 6.8 with 0.02 ml Tween-20) was sonicated (MisonixQ125, U.S.A.) for 30 min at 70 % amplitude. Thereafter, 5.0 ml of MUA solution (0.5 mM in 1:3/alcohol: H2O) was added into the mixture and kept at room temperature for 5 h for complete chemisorption of alkane thiol on the nanoparticle surface. Further, MUAnanoparticles were added to 200 mM EDC and 50 mM NHS solution and the reaction mixture was incubated for 10 minutes [28]. These NHS terminated nanoparticles were dispersed under ultrasonication (MisonixQ125, U.S.A.) at 20 °C for 10 min at 70 % amplitude. Modification of nanoparticles was confirmed by recording their Fourier Transform Infrared
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Spectra (FTIR, Alpha, Bruker, Germany) at Department of Genetics, Maharshi Dayanand University, Rohtak.
2.6. Immobilization of NR Two immobilization protocols for NR, one using nFe3O4 and the other using nZnO were developed. To achieve these, 2.0 ml NHS terminated nanoparticles were poured over the polyethylene supported semicured epoxy layer of size 5x5 cm2. The preparation was left undisturbed at room temperature for 4 hrs to allow the resin to cure fully. Thus prepared epoxy/nFe3O4 and epoxy/nZnO hybrids were used to conjugate NR. Freshly prepared enzyme solution (0.2 ml, 110 units/ml) in cold phosphate buffer (25.0 mM, pH 7.3) was first assayed for NR activity and protein concentration and then added to each of the hybrid supports individually. After 48 h at 4 °C, the resultant epoxy/nFe3O4/NR and epoxy/nZnO/NR bioconjugates were washed with distilled water until no protein was detected in the washing and assayed for NR activity. The amount of NR bound to the hybrid support was calculated as the difference between the protein content of the original enzyme solution and the amount of protein lost in all washings. The protein content was estimated by the method of Lowry et al. (1951) [29] using BSA as standard protein. Surface morphologies and bonded interactions of the epoxy/nFe3O4/NR and epoxy/nZnO/NR conjugates at successive stages of fabrication were studied using scanning electron microscopy (SEM, LEO 440) and Fourier Transform Infrared Spectroscopy (FTIR, Alpha, Bruker, Germany) respectively.
2.7. Enzymatic assay of NR NR activity was measured by determining the production of nitrite in sulfanilamide/NEDA system [30]. To determine the activity of free NR, 2.0 ml of the reaction mixture having 24.0 mM potassium phosphate pH 7.3, 0.05 mM EDTA, 9.5 mM potassium nitrate, 0.10 mM β8
NADH and 15.0 units of NR, was incubated at 30 °C for 2 min. Nitrite produced as a result of nitrate reduction by NR was measured as described by Snell and Snell (1959) [30]. The assay for immobilized NR was performed in the same way except that free enzyme was replaced by epoxy/nFe3O4/NR and epoxy/nZnO/NR preparations. One unit of NR produced 1.0 µmole of nitrite per minute in β-NADH system at 30 °C and pH 7.3.
2.8. Optimization of experimental conditions The factors such as pH, temperature and substrate concentration which may influence the activity of NR were modified to find the optimum working conditions for the enzyme. In order to judge the suitability of epoxy/nFe3O4 and epoxy/nZnO hybrids as immobilization supports, the data for immobilized NR was compared to the data acquired for free NR.
2.8.1. Effect of pH In immobilized enzymes, pH of the microenvironment significantly affects the binding of substrate and stability or solubility of the reactants and products. Hence, p H d e p e n d e n t activity profile of both the free and immobilized NR preparations was c o n s t r u c t e d using 10.0 mM acetate buffer for pH 5.0 and 5.5, 10.0 mM potassium phosphate buffer in the pH range of 6.0-8.0 and 10.0 mM borate buffer at pH 8.5 and 9.0.
2.8.2. Effect of temperature To investigate the effect of temperature on NR catalyzed reaction, the incubation temperature was varied from 5-50 °C with an interval of 5 °C. Energy of activation (Ea) was calculated from Arrhenius plot by plotting inverse of temperature (in degree Kelvin) vs. log of enzyme activity.
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2.8.3. Effect of substrate concentration Since rate of reaction catalyzed by an immobilized enzyme is significantly affected by the availability of substrate molecules in bulk as well as in close vicinity, effect of varying KNO3 and β-NADH co ncent rat i on s was st udi ed. Under opti mu m conditions of pH and temperature, KNO 3 concent ra t i on w a s var i ed from 0 . 0 1 mM to 1 3 .0 mM at a fixed β-NADH concent r at i on (0.10 mM) , wher eas concent rat i on of
β-NADH
wa s va r i ed from 0.001 to 0.15 mM at saturating KNO3 concentrations. Kinetic parameters Km and Vmax were calculated by Lineweaver-Burk plot. Vmax values were used to assess the turnover number (kcat).
2.9. Stability studies Thermal and storage stability studies were carried out both for free and immobilized enzymes, while operational stability was determined only for immobilized NR. To ascertain thermal stability, free and immobilized enzymes were exposed to t e m p e r a t u r e s r a n g i n g f r o m 25 °C to 50 °C at an interval of 5°C for 30 min and then the residual activity was measured under optimum conditions of pH, temperature and substrate concentration. Shelf life of the enzymes was determined by measuring their activity on alternate days upto 40 days, when stored in potassium phosphate buffer (25.0 mM, pH 7.0) at 4 °C. For operational stability, immobilized enzymes were repeatedly assayed at 25 °C in batch mode till the point their activity was significantly lost. After each reaction run, the immobilized enzyme preparations were washed with 10.0 mM potassium phosphate buffer (pH 7.3) to remove any residual activity.
2.10. Analytical application of immobilized NR In order to demonstrate practical usefulness of the immobilized NR preparations, epoxy/nFe3O4/NR was used for nitrate determinaiton in soil samples and epoxy/nZnO/NR
10
was used for quantification of nitrates in pond water samples. Nitrate present in the soil/water samples was reduced to nitrite using immobilized NR in the presence of β-NADH as reducing agent. Nitrite thus formed was treated with sulphanilamide, which was coupled with NEDA to form a diazocompound that gave nitrite color complex [30]. Soil samples were collected from the kitchen garden using an auger at a depth of about 20 cm. The samples were air dried, sieved and stored in labeled plastic bags. 50g of each soil sample was weighed and transferred to conical flask and was shaken with 50 ml of distilled water (1:1 ratio) for 10 minutes. The sample was left undisturbed for 30 minutes. The supernatant was filtered using Whatman no. 42 filter paper. The filtration process was repeated to remove turbidity prior to analytical determinations. To the clear samples, 0.05 mM ethylenediaminetetraaceticacid (EDTA) was added before analysis to eliminate iron, copper and other metals. Water samples from nearby ponds were collected in plastic bottles by submerging the bottles below the water level. Both soil and water samples were stored at 4 °C till use. Thus prepared/collected water samples were also analyzed for other water quality parameters such as colour, odour, pH, total dissolved solids (TDS), total hardness (TH) and dissolved oxygen (DO) at a water testing laboratory of Public Health Engineering Department, Rohtak. Nitrates in both soil and water samples were determined as described under section 2.4 for immobilized enzyme assay except that potassium nitrate solution was replaced by 0.1 ml of the water/soil samples. The immobilized enzymes were washed with 10.0 mM potassium phosphate buffer (pH 7.3) after every assay and stored in 25.0 mM potassium phosphate buffer pH 7.0 at 4 °C when not in use. Nitrate concentration in soil and water samples was intrapolated from a standard curve between sodium nitrite concentration (ranging from 0.01 to 10.0 mM) and A540.
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3. Results and discussion
3.1. Size and crystal structure of nFe3O4 and nZnO Size of nFe3O4 and nZnO was investigated using TEM images and crystal structure was confirmed by XRD. As revealed by Fig. 1A and 1C, very fine and well-dispersed nanorods of Fe3O4 as well as ZnO in the size range 6-24 nm (Fig. 1B) and 45-85 nm (Fig.1D) respectively were synthesized. Nano Fe3O4 rods were well dispersed but for nano ZnO it looked like disaggregation of some definite geometrical structure probably due to sonication of nanoparticles just before TEM analysis. The XRD pattern showing all diffraction peaks of the nFe3O4 and nZnO are shown in Fig. 2A and 2B respectively. The XRD pattern of nFe3O4 shows that the nanoparticles are polycrystalline in nature. The peak positions reveal high consistency with joint committee on powder diffraction standard (JCPDS card No. 76-1802) and are indexed to (220), (311), (400) (333) and (440), respectively [31]. It can be clearly seen from XRD diffraction pattern that synthesized iron oxide nanoparticles have high purity and good crystal quality. The dried particles exhibited very strong magnetic attraction to a magnetic rod. Well-defined peaks with high intensity in the XRD spectra suggested that nZnO particles were also of high crystallinity (Fig. 2B). The characteristic diffraction peak positions at (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) could be assigned to the crystal planes of ZnO nanorods with wurtzite crystal structures [15] and were consistent with Joint Committee for Powder Diffraction Standards (JCPDS 36-1451). Furthermore, relatively larger peak corresponding to (101) indicated that the growth of ZnO nanorods was preferentially towards plane.
3.2. Surface modification of metal oxide nanoparticles
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The thiolate group (–SH) in MUA is highly nucleophilic and has a strong affinity to the metal oxide nanoparticles [32]. MUA not only prevents aggregation of nanoparticles but also provide excellent solubility in aqueous solutions. In MUA modified nanoparticles the peripheral carboxylic groups in MUA were activated by esterification of n-hydroxysulfosuccinimide (Sulfo-NHS) catalyzed by water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) through the EDC/NHS coupling reaction. FTIR peaks corresponding to functional groups at successive stages of nanoparticles modification were recorded (Fig. 3). In FTIR spectra for bare nFe3O4 (Fig. 3A), broad absorption bands at 3289 cm-1, 1621 cm-1 and 1451 cm-1 were assigned to -OH stretching of the hydroxyl groups, H–OH bending vibration of the adsorbed water molecules and stretching regions of C-O bonds respectively (curve a) [33]. FTIR spectra for unmodified nZnO also showed appearance of similar peaks at 3319 cm-1, 1636 cm-1 and 1376 cm-1 (Fig. 3B, curve a). After modification of nanoparticles with MUA, new peaks for vibrational stretches of -CH2 groups of long alkane chains appeared at 1071 cm-1 in case of nFe3O4 (Fig. 3A, curve b) and at 1073 cm-1 and 1380 cm-1 for nZnO (Fig. 3B, curve b). This indicated successful modification of nanoparticles by MUA groups. For NHS terminated nFe3O4 (Fig. 3A, curve c) two new peaks appeared at 1830 cm-1 and 1745 cm-1. In Fig. 3B, NHS terminated nZnO (curve c) showed emergence of new peaks at 1822 cm-1 and 1748 cm-1. The new peaks at 1745 cm-1 and 1748 cm-1 were contributed by the succinimidyl carbonyl group, and peak at 1830 cm-1 and 1822 cm-1 were attributed to the band splitting of the ester carbonyl C-O stretching vibration [34]. Therefore, the difference in the peaks between bare, MUA terminated and NHS/EDC coupled nanoparticles confirmed modification/surface functionalization of both nFe3O4 and nZnO.
3.3. Immobilization of NR over epoxy/nFe3O4 and epoxy/nZnO supports
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The NHS terminated nFe3O4 and nZnO particles were affixed onto polyethylene supported epoxy layer separately to yield epoxy/nFe3O4 and epoxy/nZnO hybrid supports, which were used for immobilizing NR as shown in Scheme 1. As a result of immobilization, conjugation yield of 35.8±0.01 and 33.20±0.01 μg/cm2 with 93.72±0.50 % and 84.81±0.80 % retention of specific activity was achieved using epoxy/nFe3O4 and epoxy/nZnO supports respectively (Table 1). Hence, in terms of immobilization, epoxy/nFe3O4 support performed slightly better than epoxy/nZnO support. High yield might be attributed to larger surface area to volume ratio whereas pretty good retention of activity might be due to hydrophilic and biocompatible nature of nano particles. Nanoparticles retain a layer of bound water around them [35] which may help to preserve the activity of NR and biocompatible nature of NPs renders the immobilized enzyme “quasi free” [36]. This implies greater conformational flexibility of enzyme, which might account for high retention of activity on nanosupports. Good electrical conductivity of nanoparticles might also have facilitated more efficient electron transfer to and from the redox center of enzyme needed for efficient catalysis.
3.4. Characterization of immobilization supports by Scanning Electron Microscopy The surface morphologies of bare and modified epoxy as studied by SEM are presented in Fig. 4. SEM image of bare epoxy, revealed it to be an even and fairly smooth layer (Fig. 4A). Addition of nFe3O4 solution to partially set epoxy resin resulted in strong physical adherence of nFe3O4 to the epoxy layer (Fig. 4B) with appearance of shiny area around nanoparticles, which might correspond to scattering of charges due to the presence of NHS group on modified nFe3O4. Addition of enzyme might have neutralized the scattering, thereby decreasing the intensity of shiny halo with perceptible increase in diameter of the nanoparticles (Fig. 4C). Epoxy adhered nZnO, composed of clusters of radiating NHS modified nanorods are presented in Fig. 4D. High-density nanorods of ZnO were parallel to
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each other, gradually tapering towards the end and horizontally oriented with respect to the surface. Such an orientation is highly desirable to maximize the surface area of nanostructures. However, the absence of shiny area may be attributed to an altogether different geometrical arrangement of nZnO, a characteristic feature attributed to the polar nature of its surface. The abundance of enzyme over the surface of NHS terminated nZnO is well illustrated by Fig. 4E.
3.5. Characterization of immobilization supports by Fourier Transform Infrared Spectroscopy In order to elucidate the bonding interactions between the support and enzyme, four FTIR spectras in the range 400-4000 cm-1 were recorded for both nFe3O4 and nZnO separately i.e. of bare epoxy, epoxy/NR, epoxy/nanoparticles and epoxy/nanoparticles/NR conjugates. The FTIR spectra for bare epoxy in Fig. 5 A & B (a) revealed the presence of characteristic epoxy peaks at 830, 1033 and 1509 cm-1 for –C-O-C stretching of epoxy ring, 1, 4- substitution of aromatic ring and Ar-C=C-H stretching respectively. Variable peaks between 2915-2847 cm-1 for C-H stretching vibrations, Ar-C=C-H stretching around 1607 cm-1, bending -CH2 and CH3 asymmetrical and symmetrical between 1462-1363 cm-1, C-C-O-C- stretching at 1247 and 1183 cm-1 and aromatic ring vibrations at 719 cm-1were also present [37]. FTIR spectra of epoxy/NR (Fig. 5 A & B (b)) was similar to bare epoxy except that it showed the appearance of amide I vibrations at 1638 cm-1 associated with protein backbone conformation [38]. Peak at 557 cm-1 was characteristic of magnetic nanoparticles in Fig. 5A (c) [39], whereas peaks at 495 and 556 cm−1 in Fig. 5B (c) were ascribed to phonon absorptions of the ZnO lattice and vibration band of Zn–O bond respectively. In both Fig. 5A & B, band at 769 cm-1 may be assigned to C=O out of plane bending vibrations of aromatic rings associated with sulfo NHS ester [40]. Out of plane CH2 wagging, -O-C-O- symmetrical stretching and O-H group in plane bending were observed at 914, 1412 and 1456 cm-1 respectively [41]. The appearence of bands at 1626 and 3382 cm-1 in Fig. 5A (d) confirmed the presence of NR over
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the epoxy/nFe3O4 hybrid support. Peak at 3382 cm-1 may be contributed to hydroxyl group of the protein, whereas band at 1626 cm-1 is characteristic for amide I band in protein, which is due almost entirely to the C=O stretching vibrations of the peptide linkages [42]. Similarly, appearance of bands at 1559 and 3308 cm-1 for amide I and hydroxyl group linkages respectively in Fig. 5B (d) confirmed the presence of NR over the epoxy/nZnO hybrid support. A careful comparison of the four FTIR spectras in both Fig. 5A & 5B revealed that, characteristic peaks for epoxy were consistent in all the four curves, testifying that epoxy groups did not open upon addition of either NR or nanoparticles, which might be due to unavailability of free epoxy groups on the resin. As the resin hardens it undergoes selfpolymerization reaction involving its own epoxy groups and Bisphenol A.
3.6. Optimization of pH and temperature of free and immobilized NR Observed changes in the properties of NR after immobilization are presented in Table 2. pH dependent activity profile of NR showed that both free and epoxy/nFe3O4 bound NR were maximally active at pH 7.5, whereas epoxy/nZnO bound NR showed optimum activity at pH 7.0 (Fig. 6A). Changes in pH optima after immobilization could be interpreted in terms of the modified microenvironment and partitioning of H+ between the bulk solution and the bound enzyme molecules. Results of the present study, suggest that NR is highly pH sensitive enzyme, where even a slight decrese or increase in H+ concentrations in the vicinity of enzyme can bring about relatively large changes in its activity. Activity of free as well as both the immobilized enzymes was favoured by almost equal partition of H+ between enzyme microenvironment and bulk solutions. Though, slightly lower pH optima of NR on epoxy/nZnO than on epoxy/nFe3O4, may be attributed to the differences in their respective microenvironment, arising partly due to the differntial folding of NR on the two supports and partly due to differences in the morphology of two nanoparticles. This pH optimum is similar to that reported in literature for NR immobilization onto clay polypyrrole composite [1],
16
polypyrrole/carbon nanotubes [2], mehtyl viologen/nafion [7], laponite clay gel [10], glassy carbon disc [43] and silver [13] and gold nanoparticles [14].
Rate of NR catalyzed reaction was found to be maximum at 25 °C for both free and epoxy/n ZnO bound NR (Fig. 6B). Epoxy/nFe3O4 bound NR showed maximum activity at 30 °C. However, both the immobilized enzymes had more activity than their free counterpart beyond 30 °C. This suggests that epoxy adhered Fe3O4 and ZnO nanoparticles impart conformational stability to the enzyme at higher temperature. Activation energy (Ea) for free, epoxy/nFe3O4 and epoxy/nZnO immobilized NR was 4.37, 5.54 and 7.06 kJ/mol, respectively. Slightly higher Ea of bound enzymes is possibly ascribed to nanoparticles, which could stabilize the conformation of NR by interaction and complexation [44] and diffusional resistances encountered during the course of reaction. As the reaction proceeds and products build up, concentration gradients are established in the surroundings of bound enzyme. At this point, the reaction becomes bulk diffusion controlled and rate of reaction depends on the availability of substrate around the active site [45]. Difference in Ea values of NR on the two nanoparticles may be attributed to the differential gradients of substrate around active site and to the distinctive assembly of ZnO nanorods (Fig. 4D), wherein NR was possibly crooslinked by adjacent nanorods, thereby accounting for reduction in conformational flexibility and consequently higher Ea of bound enzyme .
3.7. Kinetic characterization A hyperbolic relationship was obtained between KNO3 concentration and NR activity upto 10.0 mM for free and epoxy/nFe3O4 bound NR and upto 11.0 mM for epoxy/nZnO bound NR, beyond which increasing KNO3 concentration had no effect on NR activity. Thus obtained saturating concentrations of 10.0 mM for free and epoxy/nFe3O4/NR and of 11.0 mM for epoxy/nZnO/NR were used for further experiments. The kinetic constants Km and 17
Vmax as calculated from Lineweaver-Burk plot are shown in Fig. 6C. Km for KNO3 was found to be lower for immobilized NR (1.54 and 1.66 mM for nFe3O4 and nZnO conjugated NR respectively) compared to the free enzyme (2.22 mM) but higher compared to the reported results for NR immobilization on pyrrole viologen (0.21 mM) [1], methyl viologen (≈ 0.25 mM) [5], laponite clay gel (0.007 mM) [10] and polyviologen (≈0.75 mM) [46]. The Vmax value for epoxy/nFe3O4 (3.33 μmol (min mg protein)-1) and epoxy/nZnO (3.84 μmol (min mg protein)-1) bound NR was slightly higher than the Vmax value of 3.13 μmol (min mg protein)-1 for free NR. Lower Km and comparatively small changes in Vmax values after immobilization indicated good substrate diffusivity and admissible changes in structure of enzyme after interaction with support. Favorable orientation of the immobilized enzyme together with the altered microenvironment and negligible diffusional restrictions might account for higher localized KNO3 concentration in the vicinity of immobilized enzyme’s active site than in the bulk solution, thereby decreasing the Km. kcat value of epoxy/nFe3O4/NR (55.83 s-1) and epoxy/nZnO/NR conjugates (65.84 s-1) was lower than that of free NR (94.84 s-1). Catalytic efficiency as judged from kcat/Km revealed that immobilization decreased the catalytic efficiency of NR, the value of kcat/Km being 36.25 and 39.66 mM-1 s-1 for epoxy/nFe3O4 and epoxy/nZnO bound NR respectively and 42.72 mM-1 s-1 for free NR. The values of kinetic constants, Km and Vmax for β-NADH were found to be 17.75, 20.31 and 24.0 μM and 3.15, 3.20 and 3.46 µmol (mg protein min)
-1
for free, epoxy/nFe3O4 and
epoxy/nZnO bound NR respectively. These observed changes in the kinetic properties may also be attributed to the distinct microenvironment of each immobilized enzyme preparation, affecting the concentrations of β-NADH in the area around active site .
3.8. Thermal stability measurements
18
The residual activity of both the immobilized enzyme preparations was determined after 30 min of incubation at temperatures ranging from 25 to 50 °C, with an interval of 5 °C. Although the activity of NR decreased with increasing temperature above 30 °C, the immobilized enzymes exhibited more temperature resistance as compared to free enzyme between 30 to 40 °C (Fig. 7A). This suggests stabilization of NR by metal oxide nanoparticles and is consistent with the results reported for galactosidase/nZnO [23], amyloglucosidase/nFe3O4 [47] and α-chymotrypsin/nFe3O4 [48] bioconjugates. The enhanced thermal stability of immobilized NR, may also be attributed to the presence of epoxy layer, which might have increased the heat absorption capacity of the support and hence the thermal stability of the enzyme [49]. However, slight difference in the thermal stability of immobilized NR preparations might be correlated with different structural conformations assumed by NR at different temperatures on the two nanosupports.
3.9. Storage stability measurements Storage stability of free and immobilized enzymes stored in potassium phosphate buffer (25.0 mM, pH 7.0) at 4 °C was measured by assaying the assay mixture daily for 48 days. Storage stability of NR on nanosupports got almost doubled as free NR was completely inactive after 20 days whereas both the immobilized enzymes observed complete loss of their activities after about 40 days (Fig. 7B). An increase in half-life of NR from 7 to 16 days for both the immobilized enzymes was achieved, which is quite encouraging as compared to the stability of NR immobilized on azure A (50 % activity lost after one day) [8], methyl viologen (50 % activity lost after two days) [8], glassy carbon disc (65 % activity lost after 3 days) [43] and clay-polypyrrole composites (80 % activity lost after 5 days) [1].
19
3.10. Analytical application Colorimetric methods for determination of nitrate contents of the agricultural soil and pond water were developed using epoxy/nFe3O4/NR and epoxy/nZnO/NR conjugates respectively. Various parameters studied to evaluate the method are presented in Table 3. Both the immobilized enzyme preparations yielded excellent results for nitrate determination and could detect nitrates as low as 0.05 mM. This minimum detection limit by the present mehtod
was
lower
compared
to
the
data
for
NR
immobilization
on
carbonnanotube/polypyrrole (0.17 mM) [2], Azure A (0.5 mM) [9] and Methyl viologen (0.1 mM) [9]. Percent recoveries of added nitrates and coefficient of variation were in agreement with
the data obtained using gold [14] and silver nanoparticles [13] immobilized NR,
indicating that the method was reproducible and reliable. To evaluate the accuracy of nitrate determination by immobilized NR, nitrate values in water and soil samples obtained by using epoxy/nFe3O4 and epoxy/nZnO bound NR showed good correlation with the values obtained by Griess method.
3.11. Physicochemical characterization of water The water samples analyzed for various physicochemical parameters were found to be colorless and odorless. Values for pH, TDS, TH and DO (Mean ± S.D., n=10) were found to be 7.48 ± 0.38, 51.3 ± 3.25 mg/L, 39.66 ± 10.50 mg/L and 7.55 ± 3.34 mg/L in the range of 7.04 – 7.70, 48.9 – 55.0 mg/L, 29.0 – 50.0 mg/L and 3.84 -10.33 mg/L respectively.
3.12. Reusability studies Reusability of immobilized NR was determined at 25 °C and pH 7.3, data for which is presented in Fig. 7C. Epoxy/nZnO bound NR was slightly more stable than epoxy/nFe3O4 bound NR. On ZnO nanosupport, the enzyme retained about 80 % and 50 % of initial activity
20
after 6 and 14 reuses respectively, whereas nFe3O4 bound NR exhibited the same retention after 5 and 10 reuses. Complete loss of activity was observed after 32 reuses for nFe3O4 and after 41 reuses on nZnO supports.
4. Conclusion Covalent immobilization of NR from Aspergillus niger onto epoxy affixed Fe3O4 and ZnO nanoparticles has been described. Out of the two supports, immobilization efficiency in terms of conjugation yield and % retention of NR activity was slighlty better for epoxy/Fe3O4 support than that for epoxy/ZnO support. However, both the supports provided a conducive microenvironment to NR, as optimum assay conditions for immobilized NR stayed close to that of free NR. Parameters which improved after immobilization were substrate affinity indicated by decreased Km values for KNO3 and stability of NR. Nanosupports significantly increased storage stability as after 16 days free enzyme retained 10 % of initial activity but both the immobilized enzymes had about 50 % of their initial activity. Thermal stability was appreciably enhanced between 30 to 40 °C. The results for soil and water nitrate determination using both the immobilized enzymes were consistent, reliable and reproducible. Reusability of immobilized NR was acceptable considering the multisubunit and complex structure of the enzyme. Overall, epoxy/nFe3O4 and epoxy/nZnO bound NR was a better analyte than free NR.
Acknowledgements Vinita Hooda is thankful to University Grant Commission (File no. 39/403-2010) and Department of Biotechnology (IPLS program) for providing financial support for this work. The authors are also thankful to Sophisticated Analytical Instrument Facility, Punjab
21
University, Chandigarh for TEM and XRD analysis; Amity University, Noida for SEM analysis and department of Genetics, Maharshi Dayanand University for FTIR analysis.
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glucose. Biosens Bioelectron 2007;23:135-139. 17 Rossi L.M., Quach A.D., Rosenzweig Z. Glucose oxidase magnetite nanoparticle bioconjugate for glucose sensing. Anal Bioanal Chem 2004;380:606-613. 18 Singh S.P., Arya S.K., Pandey P., Malhotra B.D., Saha S., Sreenivas K., Gupta V. Cholesterol biosensor based on rf sputtered zinc oxide nanoporous thin film. Appl Phys Lett 2007;91:63901-63903. 19 Kouassi G.K., Irudayaraj J., McCarty G. Examination of cholesterol oxidase attachment to magnetic nanoparticles. J Nanobiotechnol 2005;3:1-9. 20 Shang C-Y., Li W-X., Zhang R-F. Immobilized Candids antarctica lipase B on ZnO nanowires/macroporous silica composites for catalyzing chiral resolution of (R,S)-2octanol. Enzyme Microb Tech 2014;61-62:28-34. 21 Cruz-Izquierdo Á., Picó E.A., Anton-Helas Z., Boeriu C.G., Llama M.J., Serra J.L. Lipase immobilization to magnetic nanoparticles: methods, properties and applications for biobased products. N Biotechnol 2012; 29 (23-26): S100-101. 22 Husain Q., Ansari S.A., Alam F., Azam A. Immobilization of Aspergillus oryzae βgalactosidase on zinc oxide nanoparticles via simple adsorption mechanism. Int J Biol Macromol 2011;49:37-43. 23 Chen T., Yang W., Guo Y., Yuan R., Xu L., Yan Y. Enhancing catalytic performance of β-glucosidase via immobilization on metal ions chelated magnetic nanoparticles. Enzyme Microb Tech 2014;63:50-57. 24 Usman Ali S.M., Ibupoto Z.H., Salman S., Nur O., Willander M., Danielsson B. Selective determination of urea using urease immobilized on ZnO nanowires. Sensor Actuat B-Chem 2011;160(1):637-643. 25 Sahoo B., Sahu S.K., Pramanik P. A novel method for the immobilization of urease on phosphonate grafted iron oxide nanoparticle. J Mol Catal B-Enzym 2011;69(3-4):
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95-102. 26 Khatiri R., Reyhani A., Mortazavi S.Z., Hossainalipour M. Immobilization of serum albumin on the synthesized three layer core-shell structures of super-paramagnetic iron oxide nanoparticles. J Ind Eng Chem 2013;19(5):1642-1647. 27 Moghaddam A.B., Nazari T., Badraghi J., Kazemzad M. Synthesis of ZnO Nanoparticles and Electrodeposition of Polypyrrole/ZnO Nanocomposite Film. Int J Electrochem Sci 2009;4(2):247-257. 28 Li D., He Q., Cui Y., Duan L., Li J.B. Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability. Biochem Biophys Res Commun 2007;355:488-493. 29 Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the Folin Phenol Reagent. J Biol Chem 1951;193:265-275. 30 F.D. Snell, C.T.Snell, C.A. Snell, Colorimetric Methods of Analysis, third ed., Vol. IIA, Van Nostrand, New York, 1959. 31 Qu H., Caruntu D., Liu H., O’Connor C.J. Water-dispersible iron oxide magnetic nanoparticles with versatile surface functionalities. Langmuir 2011; 27:2271-2278. 32 Sunga Yun-M., Noh K., Kwaka Woo-C., Kim T.G. Enhanced glucose detection using enzyme-immobilized ZnO/ZnS core/sheath nanowires.
Sensor Actuat B
2012;161: 453-459. 33 George M.A., Hess D.W., Beck S.E., Young K.M., Roberts D.A., Vrtis R. et al. Reaction of 1,1,1,5,5,5-Hexafluoro-2,4-pentanedione (H+hfac) with iron oxide thin films. In:
Novak R.E., Rużyłło J., editors. Proceedings of the Fourth International
Symposium on Cleaning Technology in Semiconductor Device Manufacturing, New Jersey: The Electrochemical Society Inc;1996, p. 175-83. 34 Zou C., Fothergill J.C., Rowe S.W. The effect of water absorption on the dielectric
25
properties of epoxy nanocomposites, dielectrics and electrical insulation. IEEE Trans 2008;15(1):106-117. 35 Firlar E., Çınar S., Kashyap S., Akinc M., Prozorov T. Direct visualization of the hydration layer on alumina nanoparticles with the fluid cell STEM in situ. Sci Rep 2015;5:9830. 36 Ansari S.A., Husain Q. Potential applications of enzymes immobilized on/in nano materials: A review. Biotechnol Adv 2012; 30: 512-523. 37 Brito Z., Sanchez G. Influence of metallic fillers on the thermal and mechanical behaviour in composites of epoxy matrix. Compos Struct 2000;48:79-81. 38 Guerrero P., Kerry J.P., de la Caba K. FT-IR characterization of protein polysaccharide interactions in extruded blends. Carbohydr Polym 2014;111:598-605. 39 Kumar B., Smita K., Cumbal L., Debut A. Biogenic synthesis of iron oxide nanoparticles for 2-arylbenzimidazole fabrication. J Saudi Chem Soc 2014;18(4): 364-369. 40 Abdulgafour H.I., Hassan Z., Al-Hardan N., Yam F.K. Growth of zinc oxide nanoflowers by thermal evaporation method. Physica B 2010;405:2570–2572. 41 Lambert J.B., Gronert S., Shurvell H.F., Lightner D.A. Organic structural spectroscopy. 2nd ed. New York:Pearson; 2013. 42 Barth A. The infrared absorption of amino acid side chains. Prog Biophys Mol Biol 2000;74:141-73. 43 Ferreyra N.F., Solís V.M. An amperometric nitrate reductase–phenosafranin electrode:
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2004;64:61-70. 44 Wang A.M., Liu M.Q., Wang H., Zhou C., Du Z.Q., Zhu S.M., Shen S.B., Ouyang P.K. Improving enzyme immobilization in mesocellular siliceous foams by
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microwave irradiation. J Biosci Bioeng 2008;106: 286-291.
45 J. M. Engasser, C. Horvath, Diffusion and kinetics with immobilized enzymes, in: L.B. Wingard Jr., E. Katchalski-Katzir, L. Goldstein (Eds.), Applied Biochemistry and Bioengineering: Immobilized Enzyme Principles, Academic Press Inc., 1977. 46 Cosnier S., Innocent C., Jouanneau Y. Amperometric detection of nitrate via a nitrate reductase immobilized and electrically wired at the electrode surface. Anal Chem 1994;66:3198–201. 47 Gupta K., Jana A.K., Kumar S., Maiti M. Immobilization of amyloglucosidase from SSF of Aspergillus niger by cross linked enzyme aggregate onto magnetic nanoparticles using minimum amount of carrier and characterizations. J Mol Catal B-Enzym 2013;98:30-36. 48 Hong J., Gong P., Xu D., Dong L., Yao S. Stabilization of alpha-chymotrypsin by covalent immobilization on amine-functionalized superparamagnetic nanogel. J Biotechnol 2007;128:597-605. 49 Preety, Hooda V. Immobilization and kinetics of catalase on calcium carbonate nanoparticles attached epoxy support. Appl Biochem Biotechnol 2014;172:115-130.
27
Legends Scheme 1
Fabrication of epoxy affixed and NHS terminated metal oxide nanoparticles (nMOx) (A) and immobilization of NR over thus prepared nMOx (B).
Table 1
NR immobilization efficiency of epoxy/nFe3O4 and epoxy/nZnO supports.
Table 2
A comparison of the kinetic parameters of free and immobilized NR. The kinetic constants Km, Vmax and kcat/Km have been derived using KNO3 as substrate.
Table 3
Evaluation of the method for nitrate determination using epoxy/nFe3O4 and epoxy/ZnO bound NR.
Fig. 1
TEM images of nFe3O4 (A) and nZnO (C) at a scale bar of 20 and 5 nm respectively. Zoomed images show size labelled Fe3O4 (B) and ZnO (D) nanoparticles.
Fig. 2
The XRD pattern of nFe3O4 (A) and nZnO (B), showing high consistency of peak positions withjoint committee on powder diffraction standard (JCPDS card no. 76-1802 for nFe3O4 and JCPDS card no.36-1451 for nZnO).
Fig. 3
Surface modification of nFe3O4 (A) and nZnO (B) as confirmed by FTIR. Curve a shows the spectra of bare nanoparticles, curve b & c represents the changes in the spectra after addition of MUA and EDC/NHS respectively.
Fig. 4
SEM images of bare epoxy (A), epoxy/nFe3O4 (B), epoxy/nFe3O4/NR (C), epoxy/ZnO (D) and epoxy/ZnO/NR (E) conjugates at a magnification little over 30kx.
Fig. 5
FTIR spectra recorded at successive stages of NR immobilization onto epoxy/nFe3O4 (Fig. 5A) and epoxy/nZnO (Fig. 5B) hybrid supports;bare epoxy (curve a), epoxy/NR (curve b), epoxy/ nFe3O4 or nZnO (curve c) and epoxy/ nFe3O4 or nZnO/NR conjugates (curve d).
28
Fig. 6
Determination of optimum pH (A) and temperature (B) for free ( epoxy/Fe3O4 (
) and epoxy/ZnO (
),
) bound NR. Fig. 6C is showing the
Lineweaver-Burk plot relating soluble and immobilized NR reaction velocities to potassium nitrate concentrations (0 . 0 1 mM-1 3 .0 mM). Each point is a mean of three independent observations. Error bars show standard error. Fig. 7
Thermal (A), storage (B) and operational stabilities (C) of free ( ) , epoxy/nFe3O4(
) and epoxy/nZnO (
) bound NR.
29
Fig. 1.
(A)
(B)
30
(C)
(D)
31
Fig. 2
Intensity (a.u.)
(A)
Intensity (a.u.)
(B)
32
Fig. 3
(A)
(B)
33
Fig. 4
A
B
C
34
D
E
35
Fig. 5
(A)
(B) 36
Fig. 6 100
% Relative Activity
90 80 70 60 50 40 30 20 10 0 5
5.5
6
6.5
pH
7
7.5
8
8.5
9
(A)
100
% Relative Activity
90 80 70 60 50 40 30 20 10 0 5
10
15
20
25
30
35
40
45
50
Temperature (°C) (B)
37
2.8 2.4
1/V
2 1.6 1.2 0.8 0.4 0 -0.7 -0.3 0.1
0.5
0.9
1.3 1.7 2.1 1/S (mM)
2.5
2.9
3.3
3.7
4.1
(C)
38
Fig. 7
%Relative activity
120 100 80 60 40 20 0 25
30
35
40
45
50
Temperature (°C) (A)
100
%Relative activity
90 80 70 60 50 40 30 20 10 0 0
4
8
12
16
20
24
28
32
36
40
44
48
Storage time (Days) (B)
39
100 90
%Relative Activity
80 70 60 50 40 30 20 10 0 0
5
10
15
20
25
30
35
40
45
Reuse Number
(C)
40
Scheme 1:
A
nMOx
MUA/ Buffer/Tween-20
EDC/NHS
nMOx
nMOx
O
O nMOx Fe3O4 -S
OH
nMOx Fe3 O4 -C-O-N
O
O
B
NR
nMOx O4
[
]
nMOx
nMOx/NR/Conjugate
nMOx O
Modified nMOx Epoxy Layer
nMOx
Polyethylene Sheet Fe3 O4 -C-O- NnMOx
H
O
O
Fe3 O4 -C-O-N nMOx
41
O
Table 1 Support
Enzyme added to membrane (μg)
Enzyme coupled to membrane (μg)
Total activity added (units)a
% Retention Conjugation of activity yield (μg/cm2)
Epoxy/nFe3O4
200
179
22
93.72±0.50
35.8±0.01
Epoxy/nZnO
200
166±0.03
22
84.81±0.80
33.20±0.01
a
One unit of nitrate reductase (NR) is defined as the amount of NR required to reduce 1.0 µmole of nitrate into nitrite per minute in NADH system at 30 ºC and pH 7.3.
42
Table 2 Free NR
Epoxy/nFe3O4/NR conjugates
Epoxy/nZnO/NR conjugates
7.5
7.5
7.0
25
30
25
Activation energy (Ea) (kJ/mol)
4.37
5.54
7.06
K m (mM)
2.22
1.54
1.66
Vmax µmol(mg protein min)-1 kcat/Km (mM-1 s-1)
3.12
3.33
3.84
42.72 30% activity retained
36.25 55 % activity retained
39.66 33% activity retained
50% retention after 10 reuses
50% retention after 14 reuses
Kinetic parameters Optimum pH Temperature (°C)
Thermal stability at 40 º C Operational stability
--
43
Table 3 S.No.
Parameters studied
Epoxy/nFe3O4/NR
Epoxy/nZnO/NR
1.
Linearity
11.0 mM
10.0 mM
2.
Minimum detection limit
.05 mM
.05 mM
3.
Per cent recovery of added nitrate (Mean±SD, n=6) 94.82±2.12 I. 0.1mM 97.05±1.41 II. 0.2 mM
97.82±.70 96.42±1.41
Coefficient of variation (%) (n=6) 1.346 % I. Within day 3.86 % II. Between day
1.502% 2.670 %
4.
5.
Coefficient determination (n=20)
of 0.9984 (R2)
0.994
44
S0141-0229(16)30052-7 http://dx.doi.org/doi:10.1016/j.enzmictec.2016.03.007 EMT 8889
To appear in:
Enzyme and Microbial Technology
Received date: Revised date: Accepted date:
23-9-2015 6-3-2016 21-3-2016
Please cite this article as: Sachdeva Veena, Hooda Vinita.Effect of changing the nanoscale environment on activity and stability of nitrate reductase.Enzyme and Microbial Technology http://dx.doi.org/10.1016/j.enzmictec.2016.03.007 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.
Effect of changing the nanoscale environment on activity and stability of nitrate reductase
Veena Sachdeva, Vinita Hooda* 1
Department of Botany, Faculty of Life Sciences, Maharshi Dayanand University Rohtak-124001, India
*Corresponding author. Tel: +91 9896795000; fax: +91 1262 247150 Email: [email protected] 1
Postal address
1
Highlights
Nitrate reductase (NR) covalently immobilized onto epoxy affixed Fe3O4 and ZnO nanoparticles.
High immobilization efficiency with decreased Km values was achieved.
Storage and thermal stabilities improved substantially after immobilization.
Nitrate determination using immobilized NR produced reliable data.
2
Abstract Nitrate reductase (NR) is employed for fabrication of nitrate sensing devices in which the enzyme in immobilized form is used to catalyze the conversion of nitrate to nitrite in the presence of a suitable cofactor. So far, instability of immobilized NR due to the use of inappropriate immobilization matrices has limited the practical applications of these devices. Present study is an attempt to improve the kinetic properties and stability of NR using nanoscale iron oxide (nFe3O4) and zinc oxide (nZnO) particles. The desired nanoparticles were synthesized, surface functionalized, characterized and affixed onto the epoxy resin to yield two nanocomposite supports (epoxy/nFe3O4 and epoxy/nZnO) for immobilizing NR. Epoxy/nFe3O4 and epoxy/nZnO support could load as much as 35.8±0.01 and 33.20±0.01 μg/cm2 of NR with retention of about 93.72±0.50 and 84.81±0.80 % of its initial activity respectively. Changes in surface morphology and chemical bonding structure of both the nanocomposite supports after addition of NR were confirmed by scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR). Optimum working conditions of pH, temperature and substrate concentration were ascertained for free as well as immobilized NR preparations. Further, storage stability at 4 °C and thermal stability between 25 to 50 °C were determined for all the NR preparations. Analytical applications of immobilized NR for determination of soil and water nitrates along with reusability data has been included to make sure the usefulness of the procedure.
Keywords: Nitrate reductase; Zinc oxide; Iron oxide; Nanoparticles; Immobilization; Nitrate determination
3
1. Introduction Nitrate reductase (NR) is a complex multiredox center enzyme that catalyzes the reduction of nitrate to nitrite in the presence of NADH or other suitable cofactor/mediator as shown in equation (i): NO3 + NAD(P)H + H+
NO2 + NAD(P)+ + ½ H2O
----------(i)
The enzyme (NR) is usually immobilized onto conducting polymers like polypyrrole [13], alkylpyrroleviologen [4], polyviologen [5,6], methyl viologen and azure A [7-9] for construction of nitrate biosensor. Since, the redox centers of NR are deeply embedded in the protein structure, conducting polymers help to achieve better electrical wiring of enzyme to the electrode surface. On the downside, partial hydrophobic character of these immobilization matrices impose conformational constraints in the protein structure and exert diffusional restrictions within the microenvironment of bound enzyme, which greatly affects not only the activity, but also the stability of the bound enzyme [10]. Moreover, in majority of the studies, NR is weakly bonded to these matrices as immobilization is carried out by adsorption and/or entrapment. As a result, most of the nitrate biosensors perform poorly either due to denaturation of enzyme or leaching of immobilized enzyme from the polymers. Sometimes, strong bonding of NR with polymer film decreases the number of free active sites, effectively decreasing the rate of catalytic reaction [11]. Hence, the importance of a suitable immobilization matrix for stabilizing NR can no longer be ignored. Over the past few years, great interest has been generated in the use of metal and metal oxide nanoparticles for enzyme stabilization [12]. Although both metal and metal oxide nanoparticles have yielded promising results but metal oxide nanoparticles have the advantage of being more reactive due to the presence of uncoordinated atoms on their corners and edges. Earlier NR has been immobilized onto silver [13] and gold [14]
4
nanoparticles with considerable improvement in its properties, but so far it has not been immobilized onto any metal oxide nanoparticle. For this purpose, ZnO and Fe3O4 nanoparticles (nZnO and nFe3O4) are of special interest. Since nZnO has the unique ability to display different geometric nanostructures with large interfacial areas, thereby scaling up the amount of loaded enzyme and paramagnetic nature of nFe3O4 offers ease of separation under external magnetic field [15]. In addition, their biocompatible nature ensures that the loaded enzyme remains biologically active and good electrical conductivity helps in faster electron transfer to and from the active centres of enzymes. Both nZnO and nFe3O4 have so far been utilized for immobilization of a number of enzymes including glucose oxidase [16,17], cholesterol oxidase [18,19], lipase [20,21], β galactosidase [22,23] and urease [24,25] with increased activity, stability, reuse capacity and storage stability. Usually, these immobilization protocols rely on separation of nanoparticles from the solution either by centrifugation in case of nZnO or by applying external magnetic field for nFe3O4. Alternatively, the nanoparticles can be affixed onto some stable insoluble matrix such as epoxy, which will not only make the isolation of nanoparticles from solution easy but will also impart structural stability to the matrix [14]. Hence, in the present study, suitability of epoxy affixed ZnO and Fe3O4 nanoparticles (epoxy/nZnO and epoxy/nFe3O4 respectively) as immobilization supports for NR has been studied. Changes in optimum pH, optimum temperature, kinetics and stability of NR after immobilization vis a free NR are reported. Finally, analytical applications of both the immobilized NR preparations are demonstrated.
2. Materials and methods 2.1. Chemicals
5
11-mercaptoundecanoic acid (MUA), nitrate reductase (NR; NAD(P)H) from Aspergillus niger (≥ 300.0 units/g), N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), β-nicotinamide adenine dinucleotide (ß-NADH), bovine serum albumin (BSA), FeCl3·6H2O and FeCl2.4H2O were purchased from Sigma-Aldrich Co. (St. Louis, USA). N(1-naphthyl)
ethylenediaminedihydrochloride
(NEDA),
tri-sodium
citrate,
N-
hydroxysuccinimide (NHS), sulfanilamide and tween 20 were obtained from Himedia (Mumbai, India). Ethylenediaminetetraacetic acid (EDTA) and zinc nitrate were procured from Thomas Baker (Mumbai, India). Epoxy resin and bisphenol A available as a popular adhesive under the trade name “Araldite” were purchased from the local market and epoxy layer was prepared by mixing both the components in 1:1 ratio, as per the directions written on pack. All other chemicals purchased were of analytical reagent (AR) grade. Deionized water was used as solvent in all the experiments.
2.2. Synthesis of nFe3O4
Iron oxide nanoparticles were prepared by co-precipitating di and trivalent Fe ions by alkaline solution as described by Khatiri et al. (2013) [26]. FeCl3·6H2O (2.70 g) and FeCl2·4H2O (1.00 g) were dissolved into 50 mL of water. To this solution, 25 mL of 30% ammonia was added at 80 ⁰C under nonmagnetic stirring. The stirring was continued for 30 min and the reaction mixture was cooled to room temperature. The precipitate was separated by washing and centrifuging several times in water and then in ethanol at 2800 rpm. The nanoparticles were finally dried in air.
2.3. Synthesis of nZnO
To prepare nZnO, 0.45 M aqueous solution of zinc nitrate and 0.9 M aqueous solution of NaOH were prepared. The beaker containing NaOH solution was heated upto 55 °C and zinc
6
nitrate solution was added drop wise for 40 min to the heated solution under high speed stirring. The beaker was sealed at this condition for 2 h. The precipitated nZnO were cleaned with deionized water and ethanol and air-dried at 60 °C [27].
2.4. Characterization of nanoparticles Size of nanoparticles was confirmed by Transmission Electron Microscopy (TEM – Jeol 2100F) at Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi and the crystal structure was illucidated by XRD (Panalytical's X'Pert Pro) at Sophisticated Analytical Instrumentation Facility, Punjab University Chandigarh.
2.5. Surface modification and characterization of nanoparticles In order to prevent the aggregation of nanoparticles and improve their solubility, the surface of nanoparticles was first modified with MUA, a functional thiol and then MUA terminated nanoparticles were cross-linked to amino groups on the surface of enzyme using EDC/NHS coupling reaction. To prepare surface modified nanoparticles, a suspension of 5.0 mg of nanoparticles in 5.0 ml of phosphate buffer (10.0 mM, pH 6.8 with 0.02 ml Tween-20) was sonicated (MisonixQ125, U.S.A.) for 30 min at 70 % amplitude. Thereafter, 5.0 ml of MUA solution (0.5 mM in 1:3/alcohol: H2O) was added into the mixture and kept at room temperature for 5 h for complete chemisorption of alkane thiol on the nanoparticle surface. Further, MUAnanoparticles were added to 200 mM EDC and 50 mM NHS solution and the reaction mixture was incubated for 10 minutes [28]. These NHS terminated nanoparticles were dispersed under ultrasonication (MisonixQ125, U.S.A.) at 20 °C for 10 min at 70 % amplitude. Modification of nanoparticles was confirmed by recording their Fourier Transform Infrared
7
Spectra (FTIR, Alpha, Bruker, Germany) at Department of Genetics, Maharshi Dayanand University, Rohtak.
2.6. Immobilization of NR Two immobilization protocols for NR, one using nFe3O4 and the other using nZnO were developed. To achieve these, 2.0 ml NHS terminated nanoparticles were poured over the polyethylene supported semicured epoxy layer of size 5x5 cm2. The preparation was left undisturbed at room temperature for 4 hrs to allow the resin to cure fully. Thus prepared epoxy/nFe3O4 and epoxy/nZnO hybrids were used to conjugate NR. Freshly prepared enzyme solution (0.2 ml, 110 units/ml) in cold phosphate buffer (25.0 mM, pH 7.3) was first assayed for NR activity and protein concentration and then added to each of the hybrid supports individually. After 48 h at 4 °C, the resultant epoxy/nFe3O4/NR and epoxy/nZnO/NR bioconjugates were washed with distilled water until no protein was detected in the washing and assayed for NR activity. The amount of NR bound to the hybrid support was calculated as the difference between the protein content of the original enzyme solution and the amount of protein lost in all washings. The protein content was estimated by the method of Lowry et al. (1951) [29] using BSA as standard protein. Surface morphologies and bonded interactions of the epoxy/nFe3O4/NR and epoxy/nZnO/NR conjugates at successive stages of fabrication were studied using scanning electron microscopy (SEM, LEO 440) and Fourier Transform Infrared Spectroscopy (FTIR, Alpha, Bruker, Germany) respectively.
2.7. Enzymatic assay of NR NR activity was measured by determining the production of nitrite in sulfanilamide/NEDA system [30]. To determine the activity of free NR, 2.0 ml of the reaction mixture having 24.0 mM potassium phosphate pH 7.3, 0.05 mM EDTA, 9.5 mM potassium nitrate, 0.10 mM β8
NADH and 15.0 units of NR, was incubated at 30 °C for 2 min. Nitrite produced as a result of nitrate reduction by NR was measured as described by Snell and Snell (1959) [30]. The assay for immobilized NR was performed in the same way except that free enzyme was replaced by epoxy/nFe3O4/NR and epoxy/nZnO/NR preparations. One unit of NR produced 1.0 µmole of nitrite per minute in β-NADH system at 30 °C and pH 7.3.
2.8. Optimization of experimental conditions The factors such as pH, temperature and substrate concentration which may influence the activity of NR were modified to find the optimum working conditions for the enzyme. In order to judge the suitability of epoxy/nFe3O4 and epoxy/nZnO hybrids as immobilization supports, the data for immobilized NR was compared to the data acquired for free NR.
2.8.1. Effect of pH In immobilized enzymes, pH of the microenvironment significantly affects the binding of substrate and stability or solubility of the reactants and products. Hence, p H d e p e n d e n t activity profile of both the free and immobilized NR preparations was c o n s t r u c t e d using 10.0 mM acetate buffer for pH 5.0 and 5.5, 10.0 mM potassium phosphate buffer in the pH range of 6.0-8.0 and 10.0 mM borate buffer at pH 8.5 and 9.0.
2.8.2. Effect of temperature To investigate the effect of temperature on NR catalyzed reaction, the incubation temperature was varied from 5-50 °C with an interval of 5 °C. Energy of activation (Ea) was calculated from Arrhenius plot by plotting inverse of temperature (in degree Kelvin) vs. log of enzyme activity.
9
2.8.3. Effect of substrate concentration Since rate of reaction catalyzed by an immobilized enzyme is significantly affected by the availability of substrate molecules in bulk as well as in close vicinity, effect of varying KNO3 and β-NADH co ncent rat i on s was st udi ed. Under opti mu m conditions of pH and temperature, KNO 3 concent ra t i on w a s var i ed from 0 . 0 1 mM to 1 3 .0 mM at a fixed β-NADH concent r at i on (0.10 mM) , wher eas concent rat i on of
β-NADH
wa s va r i ed from 0.001 to 0.15 mM at saturating KNO3 concentrations. Kinetic parameters Km and Vmax were calculated by Lineweaver-Burk plot. Vmax values were used to assess the turnover number (kcat).
2.9. Stability studies Thermal and storage stability studies were carried out both for free and immobilized enzymes, while operational stability was determined only for immobilized NR. To ascertain thermal stability, free and immobilized enzymes were exposed to t e m p e r a t u r e s r a n g i n g f r o m 25 °C to 50 °C at an interval of 5°C for 30 min and then the residual activity was measured under optimum conditions of pH, temperature and substrate concentration. Shelf life of the enzymes was determined by measuring their activity on alternate days upto 40 days, when stored in potassium phosphate buffer (25.0 mM, pH 7.0) at 4 °C. For operational stability, immobilized enzymes were repeatedly assayed at 25 °C in batch mode till the point their activity was significantly lost. After each reaction run, the immobilized enzyme preparations were washed with 10.0 mM potassium phosphate buffer (pH 7.3) to remove any residual activity.
2.10. Analytical application of immobilized NR In order to demonstrate practical usefulness of the immobilized NR preparations, epoxy/nFe3O4/NR was used for nitrate determinaiton in soil samples and epoxy/nZnO/NR
10
was used for quantification of nitrates in pond water samples. Nitrate present in the soil/water samples was reduced to nitrite using immobilized NR in the presence of β-NADH as reducing agent. Nitrite thus formed was treated with sulphanilamide, which was coupled with NEDA to form a diazocompound that gave nitrite color complex [30]. Soil samples were collected from the kitchen garden using an auger at a depth of about 20 cm. The samples were air dried, sieved and stored in labeled plastic bags. 50g of each soil sample was weighed and transferred to conical flask and was shaken with 50 ml of distilled water (1:1 ratio) for 10 minutes. The sample was left undisturbed for 30 minutes. The supernatant was filtered using Whatman no. 42 filter paper. The filtration process was repeated to remove turbidity prior to analytical determinations. To the clear samples, 0.05 mM ethylenediaminetetraaceticacid (EDTA) was added before analysis to eliminate iron, copper and other metals. Water samples from nearby ponds were collected in plastic bottles by submerging the bottles below the water level. Both soil and water samples were stored at 4 °C till use. Thus prepared/collected water samples were also analyzed for other water quality parameters such as colour, odour, pH, total dissolved solids (TDS), total hardness (TH) and dissolved oxygen (DO) at a water testing laboratory of Public Health Engineering Department, Rohtak. Nitrates in both soil and water samples were determined as described under section 2.4 for immobilized enzyme assay except that potassium nitrate solution was replaced by 0.1 ml of the water/soil samples. The immobilized enzymes were washed with 10.0 mM potassium phosphate buffer (pH 7.3) after every assay and stored in 25.0 mM potassium phosphate buffer pH 7.0 at 4 °C when not in use. Nitrate concentration in soil and water samples was intrapolated from a standard curve between sodium nitrite concentration (ranging from 0.01 to 10.0 mM) and A540.
11
3. Results and discussion
3.1. Size and crystal structure of nFe3O4 and nZnO Size of nFe3O4 and nZnO was investigated using TEM images and crystal structure was confirmed by XRD. As revealed by Fig. 1A and 1C, very fine and well-dispersed nanorods of Fe3O4 as well as ZnO in the size range 6-24 nm (Fig. 1B) and 45-85 nm (Fig.1D) respectively were synthesized. Nano Fe3O4 rods were well dispersed but for nano ZnO it looked like disaggregation of some definite geometrical structure probably due to sonication of nanoparticles just before TEM analysis. The XRD pattern showing all diffraction peaks of the nFe3O4 and nZnO are shown in Fig. 2A and 2B respectively. The XRD pattern of nFe3O4 shows that the nanoparticles are polycrystalline in nature. The peak positions reveal high consistency with joint committee on powder diffraction standard (JCPDS card No. 76-1802) and are indexed to (220), (311), (400) (333) and (440), respectively [31]. It can be clearly seen from XRD diffraction pattern that synthesized iron oxide nanoparticles have high purity and good crystal quality. The dried particles exhibited very strong magnetic attraction to a magnetic rod. Well-defined peaks with high intensity in the XRD spectra suggested that nZnO particles were also of high crystallinity (Fig. 2B). The characteristic diffraction peak positions at (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) could be assigned to the crystal planes of ZnO nanorods with wurtzite crystal structures [15] and were consistent with Joint Committee for Powder Diffraction Standards (JCPDS 36-1451). Furthermore, relatively larger peak corresponding to (101) indicated that the growth of ZnO nanorods was preferentially towards plane.
3.2. Surface modification of metal oxide nanoparticles
12
The thiolate group (–SH) in MUA is highly nucleophilic and has a strong affinity to the metal oxide nanoparticles [32]. MUA not only prevents aggregation of nanoparticles but also provide excellent solubility in aqueous solutions. In MUA modified nanoparticles the peripheral carboxylic groups in MUA were activated by esterification of n-hydroxysulfosuccinimide (Sulfo-NHS) catalyzed by water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) through the EDC/NHS coupling reaction. FTIR peaks corresponding to functional groups at successive stages of nanoparticles modification were recorded (Fig. 3). In FTIR spectra for bare nFe3O4 (Fig. 3A), broad absorption bands at 3289 cm-1, 1621 cm-1 and 1451 cm-1 were assigned to -OH stretching of the hydroxyl groups, H–OH bending vibration of the adsorbed water molecules and stretching regions of C-O bonds respectively (curve a) [33]. FTIR spectra for unmodified nZnO also showed appearance of similar peaks at 3319 cm-1, 1636 cm-1 and 1376 cm-1 (Fig. 3B, curve a). After modification of nanoparticles with MUA, new peaks for vibrational stretches of -CH2 groups of long alkane chains appeared at 1071 cm-1 in case of nFe3O4 (Fig. 3A, curve b) and at 1073 cm-1 and 1380 cm-1 for nZnO (Fig. 3B, curve b). This indicated successful modification of nanoparticles by MUA groups. For NHS terminated nFe3O4 (Fig. 3A, curve c) two new peaks appeared at 1830 cm-1 and 1745 cm-1. In Fig. 3B, NHS terminated nZnO (curve c) showed emergence of new peaks at 1822 cm-1 and 1748 cm-1. The new peaks at 1745 cm-1 and 1748 cm-1 were contributed by the succinimidyl carbonyl group, and peak at 1830 cm-1 and 1822 cm-1 were attributed to the band splitting of the ester carbonyl C-O stretching vibration [34]. Therefore, the difference in the peaks between bare, MUA terminated and NHS/EDC coupled nanoparticles confirmed modification/surface functionalization of both nFe3O4 and nZnO.
3.3. Immobilization of NR over epoxy/nFe3O4 and epoxy/nZnO supports
13
The NHS terminated nFe3O4 and nZnO particles were affixed onto polyethylene supported epoxy layer separately to yield epoxy/nFe3O4 and epoxy/nZnO hybrid supports, which were used for immobilizing NR as shown in Scheme 1. As a result of immobilization, conjugation yield of 35.8±0.01 and 33.20±0.01 μg/cm2 with 93.72±0.50 % and 84.81±0.80 % retention of specific activity was achieved using epoxy/nFe3O4 and epoxy/nZnO supports respectively (Table 1). Hence, in terms of immobilization, epoxy/nFe3O4 support performed slightly better than epoxy/nZnO support. High yield might be attributed to larger surface area to volume ratio whereas pretty good retention of activity might be due to hydrophilic and biocompatible nature of nano particles. Nanoparticles retain a layer of bound water around them [35] which may help to preserve the activity of NR and biocompatible nature of NPs renders the immobilized enzyme “quasi free” [36]. This implies greater conformational flexibility of enzyme, which might account for high retention of activity on nanosupports. Good electrical conductivity of nanoparticles might also have facilitated more efficient electron transfer to and from the redox center of enzyme needed for efficient catalysis.
3.4. Characterization of immobilization supports by Scanning Electron Microscopy The surface morphologies of bare and modified epoxy as studied by SEM are presented in Fig. 4. SEM image of bare epoxy, revealed it to be an even and fairly smooth layer (Fig. 4A). Addition of nFe3O4 solution to partially set epoxy resin resulted in strong physical adherence of nFe3O4 to the epoxy layer (Fig. 4B) with appearance of shiny area around nanoparticles, which might correspond to scattering of charges due to the presence of NHS group on modified nFe3O4. Addition of enzyme might have neutralized the scattering, thereby decreasing the intensity of shiny halo with perceptible increase in diameter of the nanoparticles (Fig. 4C). Epoxy adhered nZnO, composed of clusters of radiating NHS modified nanorods are presented in Fig. 4D. High-density nanorods of ZnO were parallel to
14
each other, gradually tapering towards the end and horizontally oriented with respect to the surface. Such an orientation is highly desirable to maximize the surface area of nanostructures. However, the absence of shiny area may be attributed to an altogether different geometrical arrangement of nZnO, a characteristic feature attributed to the polar nature of its surface. The abundance of enzyme over the surface of NHS terminated nZnO is well illustrated by Fig. 4E.
3.5. Characterization of immobilization supports by Fourier Transform Infrared Spectroscopy In order to elucidate the bonding interactions between the support and enzyme, four FTIR spectras in the range 400-4000 cm-1 were recorded for both nFe3O4 and nZnO separately i.e. of bare epoxy, epoxy/NR, epoxy/nanoparticles and epoxy/nanoparticles/NR conjugates. The FTIR spectra for bare epoxy in Fig. 5 A & B (a) revealed the presence of characteristic epoxy peaks at 830, 1033 and 1509 cm-1 for –C-O-C stretching of epoxy ring, 1, 4- substitution of aromatic ring and Ar-C=C-H stretching respectively. Variable peaks between 2915-2847 cm-1 for C-H stretching vibrations, Ar-C=C-H stretching around 1607 cm-1, bending -CH2 and CH3 asymmetrical and symmetrical between 1462-1363 cm-1, C-C-O-C- stretching at 1247 and 1183 cm-1 and aromatic ring vibrations at 719 cm-1were also present [37]. FTIR spectra of epoxy/NR (Fig. 5 A & B (b)) was similar to bare epoxy except that it showed the appearance of amide I vibrations at 1638 cm-1 associated with protein backbone conformation [38]. Peak at 557 cm-1 was characteristic of magnetic nanoparticles in Fig. 5A (c) [39], whereas peaks at 495 and 556 cm−1 in Fig. 5B (c) were ascribed to phonon absorptions of the ZnO lattice and vibration band of Zn–O bond respectively. In both Fig. 5A & B, band at 769 cm-1 may be assigned to C=O out of plane bending vibrations of aromatic rings associated with sulfo NHS ester [40]. Out of plane CH2 wagging, -O-C-O- symmetrical stretching and O-H group in plane bending were observed at 914, 1412 and 1456 cm-1 respectively [41]. The appearence of bands at 1626 and 3382 cm-1 in Fig. 5A (d) confirmed the presence of NR over
15
the epoxy/nFe3O4 hybrid support. Peak at 3382 cm-1 may be contributed to hydroxyl group of the protein, whereas band at 1626 cm-1 is characteristic for amide I band in protein, which is due almost entirely to the C=O stretching vibrations of the peptide linkages [42]. Similarly, appearance of bands at 1559 and 3308 cm-1 for amide I and hydroxyl group linkages respectively in Fig. 5B (d) confirmed the presence of NR over the epoxy/nZnO hybrid support. A careful comparison of the four FTIR spectras in both Fig. 5A & 5B revealed that, characteristic peaks for epoxy were consistent in all the four curves, testifying that epoxy groups did not open upon addition of either NR or nanoparticles, which might be due to unavailability of free epoxy groups on the resin. As the resin hardens it undergoes selfpolymerization reaction involving its own epoxy groups and Bisphenol A.
3.6. Optimization of pH and temperature of free and immobilized NR Observed changes in the properties of NR after immobilization are presented in Table 2. pH dependent activity profile of NR showed that both free and epoxy/nFe3O4 bound NR were maximally active at pH 7.5, whereas epoxy/nZnO bound NR showed optimum activity at pH 7.0 (Fig. 6A). Changes in pH optima after immobilization could be interpreted in terms of the modified microenvironment and partitioning of H+ between the bulk solution and the bound enzyme molecules. Results of the present study, suggest that NR is highly pH sensitive enzyme, where even a slight decrese or increase in H+ concentrations in the vicinity of enzyme can bring about relatively large changes in its activity. Activity of free as well as both the immobilized enzymes was favoured by almost equal partition of H+ between enzyme microenvironment and bulk solutions. Though, slightly lower pH optima of NR on epoxy/nZnO than on epoxy/nFe3O4, may be attributed to the differences in their respective microenvironment, arising partly due to the differntial folding of NR on the two supports and partly due to differences in the morphology of two nanoparticles. This pH optimum is similar to that reported in literature for NR immobilization onto clay polypyrrole composite [1],
16
polypyrrole/carbon nanotubes [2], mehtyl viologen/nafion [7], laponite clay gel [10], glassy carbon disc [43] and silver [13] and gold nanoparticles [14].
Rate of NR catalyzed reaction was found to be maximum at 25 °C for both free and epoxy/n ZnO bound NR (Fig. 6B). Epoxy/nFe3O4 bound NR showed maximum activity at 30 °C. However, both the immobilized enzymes had more activity than their free counterpart beyond 30 °C. This suggests that epoxy adhered Fe3O4 and ZnO nanoparticles impart conformational stability to the enzyme at higher temperature. Activation energy (Ea) for free, epoxy/nFe3O4 and epoxy/nZnO immobilized NR was 4.37, 5.54 and 7.06 kJ/mol, respectively. Slightly higher Ea of bound enzymes is possibly ascribed to nanoparticles, which could stabilize the conformation of NR by interaction and complexation [44] and diffusional resistances encountered during the course of reaction. As the reaction proceeds and products build up, concentration gradients are established in the surroundings of bound enzyme. At this point, the reaction becomes bulk diffusion controlled and rate of reaction depends on the availability of substrate around the active site [45]. Difference in Ea values of NR on the two nanoparticles may be attributed to the differential gradients of substrate around active site and to the distinctive assembly of ZnO nanorods (Fig. 4D), wherein NR was possibly crooslinked by adjacent nanorods, thereby accounting for reduction in conformational flexibility and consequently higher Ea of bound enzyme .
3.7. Kinetic characterization A hyperbolic relationship was obtained between KNO3 concentration and NR activity upto 10.0 mM for free and epoxy/nFe3O4 bound NR and upto 11.0 mM for epoxy/nZnO bound NR, beyond which increasing KNO3 concentration had no effect on NR activity. Thus obtained saturating concentrations of 10.0 mM for free and epoxy/nFe3O4/NR and of 11.0 mM for epoxy/nZnO/NR were used for further experiments. The kinetic constants Km and 17
Vmax as calculated from Lineweaver-Burk plot are shown in Fig. 6C. Km for KNO3 was found to be lower for immobilized NR (1.54 and 1.66 mM for nFe3O4 and nZnO conjugated NR respectively) compared to the free enzyme (2.22 mM) but higher compared to the reported results for NR immobilization on pyrrole viologen (0.21 mM) [1], methyl viologen (≈ 0.25 mM) [5], laponite clay gel (0.007 mM) [10] and polyviologen (≈0.75 mM) [46]. The Vmax value for epoxy/nFe3O4 (3.33 μmol (min mg protein)-1) and epoxy/nZnO (3.84 μmol (min mg protein)-1) bound NR was slightly higher than the Vmax value of 3.13 μmol (min mg protein)-1 for free NR. Lower Km and comparatively small changes in Vmax values after immobilization indicated good substrate diffusivity and admissible changes in structure of enzyme after interaction with support. Favorable orientation of the immobilized enzyme together with the altered microenvironment and negligible diffusional restrictions might account for higher localized KNO3 concentration in the vicinity of immobilized enzyme’s active site than in the bulk solution, thereby decreasing the Km. kcat value of epoxy/nFe3O4/NR (55.83 s-1) and epoxy/nZnO/NR conjugates (65.84 s-1) was lower than that of free NR (94.84 s-1). Catalytic efficiency as judged from kcat/Km revealed that immobilization decreased the catalytic efficiency of NR, the value of kcat/Km being 36.25 and 39.66 mM-1 s-1 for epoxy/nFe3O4 and epoxy/nZnO bound NR respectively and 42.72 mM-1 s-1 for free NR. The values of kinetic constants, Km and Vmax for β-NADH were found to be 17.75, 20.31 and 24.0 μM and 3.15, 3.20 and 3.46 µmol (mg protein min)
-1
for free, epoxy/nFe3O4 and
epoxy/nZnO bound NR respectively. These observed changes in the kinetic properties may also be attributed to the distinct microenvironment of each immobilized enzyme preparation, affecting the concentrations of β-NADH in the area around active site .
3.8. Thermal stability measurements
18
The residual activity of both the immobilized enzyme preparations was determined after 30 min of incubation at temperatures ranging from 25 to 50 °C, with an interval of 5 °C. Although the activity of NR decreased with increasing temperature above 30 °C, the immobilized enzymes exhibited more temperature resistance as compared to free enzyme between 30 to 40 °C (Fig. 7A). This suggests stabilization of NR by metal oxide nanoparticles and is consistent with the results reported for galactosidase/nZnO [23], amyloglucosidase/nFe3O4 [47] and α-chymotrypsin/nFe3O4 [48] bioconjugates. The enhanced thermal stability of immobilized NR, may also be attributed to the presence of epoxy layer, which might have increased the heat absorption capacity of the support and hence the thermal stability of the enzyme [49]. However, slight difference in the thermal stability of immobilized NR preparations might be correlated with different structural conformations assumed by NR at different temperatures on the two nanosupports.
3.9. Storage stability measurements Storage stability of free and immobilized enzymes stored in potassium phosphate buffer (25.0 mM, pH 7.0) at 4 °C was measured by assaying the assay mixture daily for 48 days. Storage stability of NR on nanosupports got almost doubled as free NR was completely inactive after 20 days whereas both the immobilized enzymes observed complete loss of their activities after about 40 days (Fig. 7B). An increase in half-life of NR from 7 to 16 days for both the immobilized enzymes was achieved, which is quite encouraging as compared to the stability of NR immobilized on azure A (50 % activity lost after one day) [8], methyl viologen (50 % activity lost after two days) [8], glassy carbon disc (65 % activity lost after 3 days) [43] and clay-polypyrrole composites (80 % activity lost after 5 days) [1].
19
3.10. Analytical application Colorimetric methods for determination of nitrate contents of the agricultural soil and pond water were developed using epoxy/nFe3O4/NR and epoxy/nZnO/NR conjugates respectively. Various parameters studied to evaluate the method are presented in Table 3. Both the immobilized enzyme preparations yielded excellent results for nitrate determination and could detect nitrates as low as 0.05 mM. This minimum detection limit by the present mehtod
was
lower
compared
to
the
data
for
NR
immobilization
on
carbonnanotube/polypyrrole (0.17 mM) [2], Azure A (0.5 mM) [9] and Methyl viologen (0.1 mM) [9]. Percent recoveries of added nitrates and coefficient of variation were in agreement with
the data obtained using gold [14] and silver nanoparticles [13] immobilized NR,
indicating that the method was reproducible and reliable. To evaluate the accuracy of nitrate determination by immobilized NR, nitrate values in water and soil samples obtained by using epoxy/nFe3O4 and epoxy/nZnO bound NR showed good correlation with the values obtained by Griess method.
3.11. Physicochemical characterization of water The water samples analyzed for various physicochemical parameters were found to be colorless and odorless. Values for pH, TDS, TH and DO (Mean ± S.D., n=10) were found to be 7.48 ± 0.38, 51.3 ± 3.25 mg/L, 39.66 ± 10.50 mg/L and 7.55 ± 3.34 mg/L in the range of 7.04 – 7.70, 48.9 – 55.0 mg/L, 29.0 – 50.0 mg/L and 3.84 -10.33 mg/L respectively.
3.12. Reusability studies Reusability of immobilized NR was determined at 25 °C and pH 7.3, data for which is presented in Fig. 7C. Epoxy/nZnO bound NR was slightly more stable than epoxy/nFe3O4 bound NR. On ZnO nanosupport, the enzyme retained about 80 % and 50 % of initial activity
20
after 6 and 14 reuses respectively, whereas nFe3O4 bound NR exhibited the same retention after 5 and 10 reuses. Complete loss of activity was observed after 32 reuses for nFe3O4 and after 41 reuses on nZnO supports.
4. Conclusion Covalent immobilization of NR from Aspergillus niger onto epoxy affixed Fe3O4 and ZnO nanoparticles has been described. Out of the two supports, immobilization efficiency in terms of conjugation yield and % retention of NR activity was slighlty better for epoxy/Fe3O4 support than that for epoxy/ZnO support. However, both the supports provided a conducive microenvironment to NR, as optimum assay conditions for immobilized NR stayed close to that of free NR. Parameters which improved after immobilization were substrate affinity indicated by decreased Km values for KNO3 and stability of NR. Nanosupports significantly increased storage stability as after 16 days free enzyme retained 10 % of initial activity but both the immobilized enzymes had about 50 % of their initial activity. Thermal stability was appreciably enhanced between 30 to 40 °C. The results for soil and water nitrate determination using both the immobilized enzymes were consistent, reliable and reproducible. Reusability of immobilized NR was acceptable considering the multisubunit and complex structure of the enzyme. Overall, epoxy/nFe3O4 and epoxy/nZnO bound NR was a better analyte than free NR.
Acknowledgements Vinita Hooda is thankful to University Grant Commission (File no. 39/403-2010) and Department of Biotechnology (IPLS program) for providing financial support for this work. The authors are also thankful to Sophisticated Analytical Instrument Facility, Punjab
21
University, Chandigarh for TEM and XRD analysis; Amity University, Noida for SEM analysis and department of Genetics, Maharshi Dayanand University for FTIR analysis.
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27
Legends Scheme 1
Fabrication of epoxy affixed and NHS terminated metal oxide nanoparticles (nMOx) (A) and immobilization of NR over thus prepared nMOx (B).
Table 1
NR immobilization efficiency of epoxy/nFe3O4 and epoxy/nZnO supports.
Table 2
A comparison of the kinetic parameters of free and immobilized NR. The kinetic constants Km, Vmax and kcat/Km have been derived using KNO3 as substrate.
Table 3
Evaluation of the method for nitrate determination using epoxy/nFe3O4 and epoxy/ZnO bound NR.
Fig. 1
TEM images of nFe3O4 (A) and nZnO (C) at a scale bar of 20 and 5 nm respectively. Zoomed images show size labelled Fe3O4 (B) and ZnO (D) nanoparticles.
Fig. 2
The XRD pattern of nFe3O4 (A) and nZnO (B), showing high consistency of peak positions withjoint committee on powder diffraction standard (JCPDS card no. 76-1802 for nFe3O4 and JCPDS card no.36-1451 for nZnO).
Fig. 3
Surface modification of nFe3O4 (A) and nZnO (B) as confirmed by FTIR. Curve a shows the spectra of bare nanoparticles, curve b & c represents the changes in the spectra after addition of MUA and EDC/NHS respectively.
Fig. 4
SEM images of bare epoxy (A), epoxy/nFe3O4 (B), epoxy/nFe3O4/NR (C), epoxy/ZnO (D) and epoxy/ZnO/NR (E) conjugates at a magnification little over 30kx.
Fig. 5
FTIR spectra recorded at successive stages of NR immobilization onto epoxy/nFe3O4 (Fig. 5A) and epoxy/nZnO (Fig. 5B) hybrid supports;bare epoxy (curve a), epoxy/NR (curve b), epoxy/ nFe3O4 or nZnO (curve c) and epoxy/ nFe3O4 or nZnO/NR conjugates (curve d).
28
Fig. 6
Determination of optimum pH (A) and temperature (B) for free ( epoxy/Fe3O4 (
) and epoxy/ZnO (
),
) bound NR. Fig. 6C is showing the
Lineweaver-Burk plot relating soluble and immobilized NR reaction velocities to potassium nitrate concentrations (0 . 0 1 mM-1 3 .0 mM). Each point is a mean of three independent observations. Error bars show standard error. Fig. 7
Thermal (A), storage (B) and operational stabilities (C) of free ( ) , epoxy/nFe3O4(
) and epoxy/nZnO (
) bound NR.
29
Fig. 1.
(A)
(B)
30
(C)
(D)
31
Fig. 2
Intensity (a.u.)
(A)
Intensity (a.u.)
(B)
32
Fig. 3
(A)
(B)
33
Fig. 4
A
B
C
34
D
E
35
Fig. 5
(A)
(B) 36
Fig. 6 100
% Relative Activity
90 80 70 60 50 40 30 20 10 0 5
5.5
6
6.5
pH
7
7.5
8
8.5
9
(A)
100
% Relative Activity
90 80 70 60 50 40 30 20 10 0 5
10
15
20
25
30
35
40
45
50
Temperature (°C) (B)
37
2.8 2.4
1/V
2 1.6 1.2 0.8 0.4 0 -0.7 -0.3 0.1
0.5
0.9
1.3 1.7 2.1 1/S (mM)
2.5
2.9
3.3
3.7
4.1
(C)
38
Fig. 7
%Relative activity
120 100 80 60 40 20 0 25
30
35
40
45
50
Temperature (°C) (A)
100
%Relative activity
90 80 70 60 50 40 30 20 10 0 0
4
8
12
16
20
24
28
32
36
40
44
48
Storage time (Days) (B)
39
100 90
%Relative Activity
80 70 60 50 40 30 20 10 0 0
5
10
15
20
25
30
35
40
45
Reuse Number
(C)
40
Scheme 1:
A
nMOx
MUA/ Buffer/Tween-20
EDC/NHS
nMOx
nMOx
O
O nMOx Fe3O4 -S
OH
nMOx Fe3 O4 -C-O-N
O
O
B
NR
nMOx O4
[
]
nMOx
nMOx/NR/Conjugate
nMOx O
Modified nMOx Epoxy Layer
nMOx
Polyethylene Sheet Fe3 O4 -C-O- NnMOx
H
O
O
Fe3 O4 -C-O-N nMOx
41
O
Table 1 Support
Enzyme added to membrane (μg)
Enzyme coupled to membrane (μg)
Total activity added (units)a
% Retention Conjugation of activity yield (μg/cm2)
Epoxy/nFe3O4
200
179
22
93.72±0.50
35.8±0.01
Epoxy/nZnO
200
166±0.03
22
84.81±0.80
33.20±0.01
a
One unit of nitrate reductase (NR) is defined as the amount of NR required to reduce 1.0 µmole of nitrate into nitrite per minute in NADH system at 30 ºC and pH 7.3.
42
Table 2 Free NR
Epoxy/nFe3O4/NR conjugates
Epoxy/nZnO/NR conjugates
7.5
7.5
7.0
25
30
25
Activation energy (Ea) (kJ/mol)
4.37
5.54
7.06
K m (mM)
2.22
1.54
1.66
Vmax µmol(mg protein min)-1 kcat/Km (mM-1 s-1)
3.12
3.33
3.84
42.72 30% activity retained
36.25 55 % activity retained
39.66 33% activity retained
50% retention after 10 reuses
50% retention after 14 reuses
Kinetic parameters Optimum pH Temperature (°C)
Thermal stability at 40 º C Operational stability
--
43
Table 3 S.No.
Parameters studied
Epoxy/nFe3O4/NR
Epoxy/nZnO/NR
1.
Linearity
11.0 mM
10.0 mM
2.
Minimum detection limit
.05 mM
.05 mM
3.
Per cent recovery of added nitrate (Mean±SD, n=6) 94.82±2.12 I. 0.1mM 97.05±1.41 II. 0.2 mM
97.82±.70 96.42±1.41
Coefficient of variation (%) (n=6) 1.346 % I. Within day 3.86 % II. Between day
1.502% 2.670 %
4.
5.
Coefficient determination (n=20)
of 0.9984 (R2)
0.994
44