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Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
INFLUENCE OF pH AND TEMPERATURE ON THE ACTIVITY OF SnO2-BOUND αAMYLASE: A GENOTOXICITY ASSESSMENT OF SnO2 NANOPARTICLES Mohd Jahir Khan
a b
& Qayyum Husain
a c
a
Department of Biochemistry , Faculty of Life Sciences, Aligarh Muslim University , Aligarh , India b
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School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor Darul Ehsan , Malaysia c
College of Medical Sciences, Jazan University , Jazan, Kingdom of Saudi Arabia Accepted author version posted online: 02 Sep 2013.Published online: 05 Feb 2014.
To cite this article: Mohd Jahir Khan & Qayyum Husain (2014) INFLUENCE OF pH AND TEMPERATURE ON THE ACTIVITY OF SnO2-BOUND α-AMYLASE: A GENOTOXICITY ASSESSMENT OF SnO2 NANOPARTICLES, Preparative Biochemistry and Biotechnology, 44:6, 558-571, DOI: 10.1080/10826068.2013.835732 To link to this article: http://dx.doi.org/10.1080/10826068.2013.835732
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Preparative Biochemistry & Biotechnology, 44:558–571, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.835732
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INFLUENCE OF pH AND TEMPERATURE ON THE ACTIVITY OF SnO2-BOUND a-AMYLASE: A GENOTOXICITY ASSESSMENT OF SnO2 NANOPARTICLES
Mohd Jahir Khan1,2 and Qayyum Husain1,3 1 Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India 2 School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor Darul Ehsan, Malaysia 3 College of Medical Sciences, Jazan University, Jazan, Kingdom of Saudi Arabia
& Immobilization of biologically important molecules on a myriad of nanosized materials has attracted great attention due to their small size, biocompatibility, higher surface-to-volume ratio, and lower toxicity. These properties make nanoparticles (NPs) a superior matrix over bulk material for the immobilization of enzymes and proteins. In the present study, Bacillus amyloliquefaciens a-amylase was immobilized on SnO2 nanoparticles by a simple adsorption mechanism. Nanoparticle-adsorbed enzyme retained 90 % of the original enzyme activity. Thermal stability of nanosupport was investigated by thermogravimetric and differential thermal analysis. Scanning electron microscopic studies showed that NPs have porous structure for the high-yield immobilization of a-amylase. The genotoxicity of SnO2-NPs was analyzed by pUC19 plasmid nicking and comet assay and revealed that no remarkable DNA damage occurred in lymphocytes. The pH-optima was found to be the same for both free and SnO2-NPs bound enzyme, while the temperature-optimum for NPs-adsorbed a-amylase was 5 C higher than its free counterpart. Immobilized enzyme retained more than 70% enzyme activity even after its eight repeated uses. Keywords a-amylase, genotoxicity, immobilization, SnO2-NPs, starch hydrolysis
INTRODUCTION Amylases are industrially important enzymes, catalyzing the hydrolysis of starch, glycogen, and other polysaccharides into small molecules.[1] These enzymes are applied in the production of bioethanol and of high fructose and maltose syrups, as well as synthesis of many pharmaceuticals.[2,3] Since the opening of new avenues in the field of biotechnology, Address correspondence to Qayyum Husain, College of Medical Sciences, Jazan University, Jazan, Post Box-114, Kingdom of Saudi Arabia. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/lpbb.
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the spectrum of amylase applications has been expanded into several fields, like clinical, medicinal, and analytical chemistry.[4] Amylases are ubiquitous in nature, are produced by bacteria, yeast, fungi, higher plants, and animals, and play a dominant role in carbohydrate metabolism. In spite of their wide distribution, bacterial amylases are preferred for industrial purposes over the others due to cost-effectiveness, easy production, and simple processing, modification, and optimization.[5] Immobilization of enzyme is important not only for its reusability but also for effective industrial uses. Immobilized enzymes are more robust and resistant to environmental changes as compared to free enzymes.[6] Hence, different immobilization techniques and supports have been investigated and utilized for several years to enhance activity, lifetime, and stability of the commercially useful enzymes like amylases.[7–11] Nanotechnology is now being applied in several fields including biotechnology, physics, chemistry, optics, and mechanics, and it is considered a trigger of the next industrial revolution by many contemporary experts.[12,13] The interaction of protein with the planar surfaces of bulk materials often induces considerable changes in their secondary and tertiary structure, while the high curvature of nanoparticles (NPs) protects their native structure.[14,15] Furthermore, metal NPs have a higher isoelectric point that is suitable for the absorption of proteins with lower isoelectric point, as immobilization is primarily driven by electrostatic interaction.[16] These properties make nanomaterials superior supports as compared to the traditional bulk materials for the immobilization of enzyme and other biomolecules.[17,18] In this study, the immobilization of a-amylase on SnO2-NPs is evaluated simply by adsorption. The surface morphology of NPs before and after binding with enzyme is characterized by scanning electron microscopy (SEM). Thermal stability of SnO2-NPs are studied with thermogravimetric analysis (TGA) and differential thermal analysis (DTA). Genotoxicity of NPs is examined by pUC19 plasmid nicking and comet assay. Immobilized a-amylase is also explored for its applicability and efficiency, including recyclability and stability.
MATERIALS AND METHODS a-Amylase (Bacillus amyloliquefaciens; EC 3.2.1.1) was purchased from Sigma (St. Louis, MO). Starch, maltose, glucose, and 3,5-dinitro-salicylic acid (DNS) were obtained from SRL Chemicals (Mumbai, India). All other chemicals and reagents were of analytical grade and used without further purification.
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Synthesis and Characterization of Nanoparticles
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SnO2-NPs were synthesized and characterized according to the procedure described in our previous study.[19] In short, a sol solution was prepared by dissolving 3.0 g of SnCl4 5H2O in 100 mL methanol under vigorous stirring. Aqueous ammonia solution (4.0 mL) was added drop wise to this solution. The resulting gel was filtered and washed with methanol to remove impurities and dried over 80 C for 5 hr in order to remove water. Finally, dried gel powder was calcined at 400 C for 2 hr, which resulted in formation of SnO2-NPs.
Adsorption of a-amylase on SnO2-NPs SnO2-NPs (100 mg) were added into a-amylase (1820 U) containing 50 mM phosphate buffer, pH 6.0. The immobilization was carried out overnight in a shaking water bath at 25 C. After immobilization, enzyme-bound NPs were collected by centrifugation at 5,000 g for 20 min at 4 C and washed thrice with the same buffer to remove unbound enzyme. Finally, immobilized enzyme was stored in assay buffer at 4 C for further study.
Assay of a-amylase The activity of a-amylase was measured spectrophotometrically by determining the amount of reducing groups formed after enzymatic hydrolysis.[20] Assay solutions contained an appropriate amount of free= immobilized enzyme in starch solution (1.0%, w=v). The reaction mixture was incubated at 50 C for 10 min in a shaking water bath. Reaction was stopped by adding 1.0 mL of DNS reagent and tubes were kept in boiling water for 5 min to develop color. The reaction mixture was diluted with distilled water, and absorbance was recorded by double-beam spectrophotometer at 540 nm. One unit of a-amylase activity is defined as the amount of enzyme that catalyzes the production 1.0 mmol of reducing groups per minute under the standard assay conditions.
Scanning Electron Microscopy The surface characteristics of SnO2-NPs and enzyme-immobilized SnO2-NPs were studied with SEM (Zeiss EVO 50, Germany). To avoid any surface charging, NPs were coated with the film of Au-Pd using a sputter coater. The specimens were examined at 20 kV accelerating voltage.
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TGA and DTA of SnO2-NPs TGA and DTA were applied to investigate the thermal properties and stability of nanosupport in N2. The experiment was performed at a heating rate of 10 C=min with a Mettler-3000 thermal analyzer and TA-Q2000 DSC for TGA and DTA, respectively.
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Genotoxicity Analysis of SnO2-NPs by Plasmid Nicking Assay Conversion of supercoiled plasmid DNA into open circular and further linear form has been used as an index of DNA breakage. A single-strand breaks in DNA can be determined by electrophoresis via differential mobility of supercoiled, open circle, and linear forms of the plasmid. The assay was performed with pUC19 plasmid DNA as described by Kitts et al with minor modifications.[21] Reactions were carried out using 1.0 mg of pUC19 DNA with 50 and 100 mg=mL of NPs in 20 mM Tris-HCl, pH 7.5, at room temperature for 2 hr prior to addition of 10 mL gel loading buffer. The control and treated DNA solutions were loaded on agarose gel and electrophoresis was performed in TBE running buffer. Finally, gel was visualized on an ultraviolet (UV) transilluminator and imaged with a gel documentation system. Alkaline Comet Assay of SnO2-NPs Comet assay (single-cell gel electrophoresis) was performed to examine the genotoxicity of NPs. The result is based on the migration of nuclear DNA to form a tail-like structure. Assay was performed according to the procedure of Dhawan et al. with some modifications.[22] Microscopic slides were coated with 1.0% normal-melting-point (NMP) agarose and allowed to air dry. A mixture of NPs-treated cells (lymphocytes) with 80 mL of 1.0% low-melting-point (LMP) agarose was added to the slides, which were immediately covered with cover slips and kept for 10 min in a refrigerator to solidify. After solidification, cover slips were removed gently and slides were immersed in cold lysing solution (2.5 mM NaCl, 1% Triton X-100, 10% dimethyl sulfoxide [DMSO], 10 mM ethylenediamine tetraacetic acid [EDTA], 10% sodium lauroyl sarcosinate, and 100 mM Tris, pH 10) for 1 hr at 4 C. DNA was kept for 30 min in freshly prepared alkaline electrophoresis solution (30 mM NaOH and 1.0 mM EDTA, pH > 13). Electrophoresis was performed at 4 C in 300 mA current. Afterward, slides were gently immersed in neutralization buffer (0.4 M Tris-HCl, pH 7.5) for 10 min, before a final wash with deionized water and covering with cover slips. Slides were scored with image analysis system (Comet 5.5; Kinetic Imaging, Liverpool, UK) attached to an Olympus fluorescent microscope
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(CX 41, Olympus Optical Randomly selected images DNA damage. Migration micrometer by a Comet 5.5
Co., Tokyo, Japan) at 200 magnification. were analyzed from each sample to assess of DNA from nucleus was measured in image analysis system.
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Influences of pH and Temperature on the Activity of a-amylase The pH optima for free and immobilized enzyme were evaluated by determining the activity of enzyme in buffers of different pH (4.0–8.0) values using different buffers. Molarity of each buffer was 50 mM and the percent maximum activity at pH 6.0 was taken as control (100%) for the calculation of remaining percent activity for soluble and SnO2-NPs bound a-amylase. The temperature optima of soluble and immobilized a-amylase were determined at varying temperatures over the range 20–80 C in a standard buffer (50 mM, pH 6.0). The enzyme activity at 50 C and that at 55 C were taken as control (100%) for calculation of residual percent activity of soluble and immobilized a-amylase, respectively. Reusability of SnO2-NPs Bound a-amylase Reusability of SnO2-NPs bound a-amylase was examined at optimum pH and temperature. After each run, enzyme-bound NPs were separated, washed with assay buffer, then reintroduced into fresh reaction medium. This procedure was repeated for 8 successive days and the activity determined on the first day was considered as control (100%) for calculating residual activity after each repeated use. Estimation of Protein Protein concentration was determined by the protein–dye binding method.[23] Bovine serum albumin was used as the standard protein. RESULTS AND DISCUSSION Immobilization Efficiency of SnO2-NPs-Bound a-amylase Nanoparticles exhibited interesting properties, such as large surface area, high catalytic efficiency, and strong adsorption ability, that make them potential candidates for enzyme immobilization. Nanomaterials strongly bind to several molecules, such as chemical compounds, drugs, probes, and proteins, either covalently or by adsorption.[24,25] Table 1 shows the immobilization efficiency of Bacillus amyloliquifaciens a-amylase on
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Genotoxicity Assessment of SnO2 Nanoparticles TABLE 1
a-Amylase Immobilized on SnO2-NPs Activity Bound to SnO2-NPs (U)
Enzyme Loaded (X) (U)
Enzyme Activity in Washes (Y) (U)
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1820
1132
Theoretical (X – Y ¼ A)
Actual (B)
Effectiveness Factor (B=A) ¼ (g)
Activity Yield % (B=A 100)
621
0.90
90%
688
SnO2-NPs by physical adsorption. NPs-adsorbed enzyme retained 90% enzyme activity, which is significantly higher than the earlier reported work. The preserved activity of a-amylase was 86% and 26–47% when covalently immobilized onto poly(2-hydroxyethyl methacrylate) and polyaniline supports, respectively.[26,27] Furthermore, 87% activity yield has been reported for Penicillium griseofulvum a-amylase adsorbed onto Celite.[28] SEM Characterization The binding of a-amylase with SnO2-NPs was investigated by SEM analysis. The result showed that NPs have fine and uniform structure with some agglomerates to form bigger clusters (Figure 1a). After immobilization, surfaces of NPs become rough and covered with enzymes (Figure 1b). SEM images demonstrate that the fine particles were the major population, providing large surface area and porous structure for loading the significant amount of enzyme. TGA and DTA Figure 2 exhibits the thermostability of SnO2-NPs by TGA and DTA. The TGA curve demonstrates that the weight loss at 125 C was 2.7%, which is attributed to removal of water=solvent mixture from the sample (Figure 2a). Additional weight loss (3.4%) was observed at 600 C representing the removal of solvent molecules that were entrapped in the porous SnO2 matrix. DTA study showed that thermal decomposition of the SnO2-NPs begins at a temperature higher than 300 C in air under atmospheric pressure (Figure 2b). Genotoxicity Assessment of SnO2-NPs Genotoxicity is considered one of the most important toxic endpoints in most chemical toxicity testing and risk assessment. The toxicity evaluation of SnO2-NPs is very important if we are going to use them in the food
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FIGURE 1 Scanning electron microscopy for free and enzyme bound SnO2-NPs. Free and aamylase-bound SnO2-NPs were coated with Au-Pd to minimize surface charging. The specimens were observed at 20 kV accelerating voltage and 40k magnification. SEM micrographs show SnO2-NPs (a) and enzyme-bound SnO2-NPs (b).
and pharmaceutical industry. In this study, the genotoxicity of SnO2-NPs was investigated by plasmid nicking and comet assay. Plasmid nicking assay representing the breakage in plasmid DNA was negligible at 100 mg=mL of NPs. The band intensities of the treated samples were almost similar to the control sample. This confirms that SnO2-NPs are not toxic up to 100 mg=mL concentrations (Figure 3). The genotoxicity was further analyzed by comet assay where the extent of DNA migration is directly related to its degradation in lymphocytes.[29] The result showed that the DNA breaking was insignificant up to
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FIGURE 2 Thermogravimetric and differential thermal analysis of nanosupport. TGA (a) was performed with a Mettler 3000 thermal analyzer using 2 mg sample with heating rate of 10 C=min in N2 atmosphere. DTA (b) was also carried in similar heating range by using TA-Q2000, DSC.
100 mg=mL of NPs (Figure 4A). In Figure 4B, the histogram represents the percentage DNA in tail of the comet since tail length is an index of DNA damage. The result showed that no remarkable changes in the tail length were observed at different concentrations of SNO2-NPs. It has been reported that the genotoxicity of NPs is either from direct interaction of NPs with DNA or from release of toxic ions from soluble NPs. Furthermore, generation of oxidative stress is also an important factor that may cause the degradation of DNA in lymphocytes.[30] In a similar study, Jin et al. reported that silica nanocomposites have no significant toxic effects at the molecular and cellular levels below a concentration of 0.1 mg=ml.[31]
Optimum pH and Temperature for the Free and NPs-Bound a-Amylase Stability of enzymes against denaturing agents is an important factor for industrial exploitation. The influence of pH on the activity of soluble and SnO2-NPs adsorbed a-amylase was studied over a range of pH, 4.0–8.0 (Figure 5). The optimum pH for starch hydrolysis was pH 6.0 for both free and adsorbed enzymes. However, the immobilized a-amylase exhibited
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FIGURE 3 Plasmid nicking assay of SnO2-NPs. Toxicity of SnO2-NPs was analyzed by plasmid nicking assay as described in the text. Circular double-stranded pUC19 DNA was used in this study. Lane (C), control pUC19 DNA; (T1) pUC19 DNA treated with 50 mg=mL SnO2-NPs; (T2) pUC19 DNA treated with 100 mg=mL SnO2-NPs.
significant broadening in pH-activity profile as compared to its soluble counterpart. Immobilized a-amylase retained 61% activity at pH 8.0, while soluble enzyme exhibited only 40% activity under similar experimental conditions. The observed higher stability of immobilized a-amylase against pH might be due to binding of enzyme with nanosupport, which prevents the unfolding=denaturation of enzyme.[32] It was presumed that the configuration of a-amylase was fixed on the surface of SnO2-NPs, which may increase the enzyme’s tolerability of pH variance in the surrounding microenvironment. Figure 6 shows the effect of temperature on the activity of soluble and immobilized a-amylase. Soluble enzyme has an optimum temperature of 50 C, whereas the maximum activity of immobilized a-amylase was found at 55 C. Initially, the catalytic activity of free enzyme was enhanced with increasing temperature up to the optimum temperature, followed by a
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FIGURE 4 (A) Comet assay of SnO2-NPs. Quantitative analysis of nuclear DNA damage was observed by Comet assay as given in the text. The microscopic images have been shown for control DNA (C), DNA treated with 50 mg=mL SnO2-NPs (T1), and 100 mg=mL SnO2-NPs (T2). (B) Comet assay histogram of SnO2-NPs. Histogram shows control DNA (C) treated with 50 mg=mL SnO2-NPs (T1) and with 100 mg=mL SnO2-NPs (T2).
FIGURE 5 pH–activity profiles for soluble and immobilized a-amylase. The activity of soluble and immobilized a-amylase was measured in buffers (50 mM) of various pH (pH 4.0–8.0). The molarity of each buffer was 50 mM. The activity at pH 6.0 was taken as control (100%) for calculating remaining percent activity for both enzyme preparations at different pH.
rapid decline in the activity of free enzyme as compared to the immobilized a-amylase. This might be because of adsorption of the enzyme on the support restricting its free movement, providing extended stability even at higher temperatures. Therefore, the enzyme might have higher activation
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FIGURE 6 Temperature–activity profiles for soluble and immobilized a-amylase. The activity for soluble and immobilized a-amylase was assayed at various temperatures (40–80 C). The enzyme activity at 50 C and 55 C was taken as control for calculating remaining percent activity for soluble and NPs bound aamylase, respectively.
FIGURE 7 Reusability of immobilized a-amylase. The reusability of SnO2-NPs bound a-amylase was monitored for 8 successive days. The activity determined on the first day was taken as control (100%) to calculate remaining percent activity after each repeated use.
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energy to show its catalytic activity. Similar results were shown by earlier investigators.[33,34]
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Reusability One of the major hurdles for cost effectiveness of enzymatic processes is the inability of reuse the enzyme in a particular reaction. The immobilized a-amylase can be used repeatedly for starch hydrolysis. It was found that immobilized enzyme was active, holding over 90% activity after 4 repeated cycles and 77% activity even after 8 cycles of use (Figure 7). Thus, the current study agrees with the fact that entailing structural stabilization of immobilized enzyme minimizes the possibility of leaching of enzyme from the support and shows better activity and efficacy.
CONCLUSIONS SnO2-NPs structure provided a favorable microenvironment for the immobilization of a-amylase. The SnO2-NPs-bound enzyme not only broadened its pH optimum but also showed an elevated temperature optimum, overcoming the problems related to the gelatinization of starch during hydrolysis with significantly decreased possible microbial contamination. The genotoxicity study further supports that SnO2-NPs-bound enzyme can be safely used in food industries as well as in health sector. Thus, the system containing SnO2-NPs-adsorbed a-amylase can be exploited for the hydrolysis of starch in batch as well as in continuous systems.
ACKNOWLEDGMENTS The authors gratefully acknowledge EM Central Facility, Textile Technology Department, IIT Delhi, India, for SEM analysis. The authors are also thankful to the University Grants Commission, New Delhi, India, for sponsoring a fellowship to one of us (M. J. Khan).
REFERENCES 1. Ben Abdelmalek-Khedher, I.; Urdaci, M.C.; Limam, F.; Schmitter, J.M.; Marzouki, M.N.; Bressollier, P. Purification, Characterization and Partial Primary Sequence of a Major-Maltotriose-Producing a-Amylase, ScAmy43, From Sclerotinia sclerotiorum. J. Microb. Biotechnol. 2008, 18, 1555–1563. 2. Sharma, A.; Satyanarayana, T. Optimization of Medium Components and Cultural Variables for Enhanced Production of Acidic High Maltose-Forming and Ca2þ-Independent a-Amylase by Bacillus acidicola. J. Biosci. Bioeng. 2011, 111, 550–553.
Downloaded by [Florida International University] at 21:21 05 January 2015
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M. J. Khan and Q. Husain
3. Sivaramakrishnan, S.; Gangadharan, D.; Nampoothiri, K.M.; Soccol, C.R.; Pandey, A. a-Amylases From Microbial Sources: An Overview on Recent Developments. Food Technol. Biotechnol. 2006, 44, 173–184. 4. Gupta, R.; Gigras, P.; Mohapatra, H.; Goswami, V.K.; Chauhan, B. Microbial a-Amylases: A Biotechnological Perspective. Process Biochem. 2003, 38, 1599–1616. 5. Pandey, A.; Nigam, P.; Soccol, C.R.; Soccol, V.T.; Singh, D.; Mohan, R. Advances in Microbial Amylases. Biotechnol. Appl. Biochem. 2000, 31, 135–152. 6. Khan, M.J.; Husain, Q.; Ansari, S.A. Polyaniline-Assisted Silver Nanoparticles: A Novel Support for the Immobilization of a-Amylase. Appl. Microbiol. Biotechnol. 2013, 97, 1513–1522. 7. Kahraman, M.V.; Bayramoglu, G.; Kayaman-Apohan, N.; Gungor, A. a-Amylase Immobilization on Functionalized Glass Beads by Covalent Attachment. Food Chem. 2007, 104, 1385–1392. 8. Tee, B.L.; Kaletunc, G. Immobilization of a Thermostable a-Amylase by Covalent Binding to an Alginate Matrix Increases High Temperature Usability. Biotechnol. Prog. 2009, 25, 436–445. 9. Cordeiro, A.L.; Lenk, T.; Werner, C. Immobilization of Bacillus licheniformis a-Amylase Onto Reactive Polymer Films. J. Biotechnol. 2011, 154, 216–221. 10. Yang, K.; Xu, N.S.; Su, W.W. Co-Immobilized Enzymes in Magnetic Chitosan Beads for Improved Hydrolysis of Macromolecular Substrates Under a Time-Varying Magnetic Field. J. Biotechnol. 2010, 148, 119–127. 11. Sun, Y.M.; Chen, J.P.; Chu, D.H. Preparation and Characterization of a-Amylase Immobilized Thermal Responsive Composite Hydrogel Membranes. J. Biomed. Mater. Res. 1999, 45, 125–132. 12. Salata, O.V. Applications of Nanoparticles in Biology and Medicine. J. Nanobiotechnol. 2004, 2, 1–6. 13. Ansari, S.A.; Husain, Q. Potential Applications of Enzymes Immobilized on=in Nano Materials: A Review. Biotechnol. Adv. 2012, 30, 512–523. 14. Li, J.; Wang, J.; Gavalas, V.G.; Atwood, D.A.; Bachas, L.G. Alumina-Pepsin Hybrid Nanoparticles With Orientation-Specific Enzyme Coupling. Nano Lett. 2003, 3, 55–58. 15. Chow, D.C.; Johannes, M.S.; Lee, W.-K.; Clark, R.L.; Zauscher, S.; Chilkoti, A. Nanofabrication With Biomolecules. Mater. Today 2005, 8, 30–39. 16. Grealis, C.; Magner, E. The Oxidation of Cytochrome c in Nonaqueous Solvents. Chem. Commun. 2002, 8, 816–817. 17. Topoglidis, E.; Campbell, C.J.; Cass, A.E.G.; Durrant, J.R. Factors That Affect Protein Adsorption on Nanostructured Titania Films. A Novel Spectroelectrochemical Application to Sensing. Langmuir 2001, 17, 7899–7906. 18. Khan, M.J.; Husain, Q.; Azam, A. Immobilization of Porcine Pancreatic a-Amylase on Magnetic Fe2O3 Nanoparticles: Applications to the Hydrolysis of Starch. Biotechnol. Bioprocess. Eng. 2012, 17, 377–384. 19. Khan, M.J.; Qayyum, S.; Alam, F.; Husain, Q. Effect of Tin Oxide Nanoparticle Binding on the Structure and Activity of a-Amylase From Bacillus amyloliquefaciens. Nanotechnology 2011, 22, 455708– 455715. 20. Bernfeld, P. Amylase, a and b. In Methods in Enzymology. Colowick, S.P., Kaplan, N.O., Eds.; Academic Press, New York, NY, 1955; p. 149. 21. Kitts, D.D.; Wijewickreme, A.N.; Hu, C. Antioxidant Properties of a North American Ginseng Extract. Mol. Cell. Biochem. 2000, 203, 1–10. 22. Dhawan, A.; Bajpayee, M.; Parmar, D. Comet Assay: A Reliable Tool for the Assessment of DNA Damage in Different Models. Cell. Biol. Toxicol. 2009, 25, 5–32. 23. Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein–Dye Binding. Anal. Biochem. 1976, 72, 248–254. 24. Chen, B.; Pernodet, N.; Rafailovich, M.H.; Bakhtina, A.; Gross, R.A. Protein Immobilization on Epoxy-Activated Thin Polymer Films: Effect of Surface Wettability and Enzyme Loading. Langmuir 2008, 24, 13457–13464. 25. Luo, X.L.; Xu, J.J.; Du, Y.; Chen, H.Y. A Glucose Biosensor Based on Chitosan-Glucose Oxidase-Gold Nanoparticles Biocomposite Formed by One-Step Electrodeposition. Anal. Biochem. 2004, 334, 284–289. 26. Arica, M.Y.; Hasirci, V.; Alaeddinoglu, N.G. Covalent Immobilization of a-Amylase Onto pHEMA Microspheres: Preparation and Application to Fixed Bed Reactor. Biomaterials 1995, 16, 761–768. 27. Ashly, P.C.; Joseph, M.J.; Mohanan, P.V. Activity of Diastase a-Amylase Immobilized on Polyanilines (PANIs). Food Chem. 2011, 127, 1808–1813.
Downloaded by [Florida International University] at 21:21 05 January 2015
Genotoxicity Assessment of SnO2 Nanoparticles
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28. Ertan, F.; Yagar, H.; Balkan, B. Some Properties of Free and Immobilized a-Amylase From Penicillium griseofulvum by Solid State Fermentation. Prep. Biochem. Biotechnol. 2006, 36, 81–91. 29. Collins, A.R.; Oscoz, A.A.; Brunborg, G.; Gaivao, I.; Giovannelli, L.; Kruszewski, M.; Smith, C.C.; Stetina, R. The Comet Assay: Topical Issues. Mutagenesis 2008, 23, 143–151. 30. Moreira, S.; Silva, N.B.; Almeida-Lima, J.; Rocha, H.A.; Medeiros, S.R.; Alves, C., Jr.; Gamma, F.M. BC Nanofibres: In Vitro Study of Genotoxicity and Cell Proliferation. Toxicol. Lett. 2009, 189, 235–241. 31. Jin, Y.; Kannan, S.; Wu, M.; Zhao, J.X. Toxicity of Luminescent Silica Nanoparticles to Living Cells. Chem. Res. Toxicol. 2007, 20, 1126–1133. 32. Varavinit, S.; Chaokasem, N.; Shobsngo, S. Immobilization of a Thermostable a-Amylase. Sci. Asia 2002, 28, 247–251. 33. Ravian, P.; Tang, J.; Rasco, B.A. Thermal Stability of a-Amylase From Aspergillus oryzae Entrapped in Polyacrylamide Gel. J. Agric. Food Chem. 2003, 51, 5462–5466. 34. Kumar, R.S.S.; Vishwanath, K.S.; Singh, S.A.; Rao, A.G.A. Entrapment of a-Amylase in Alginate Beads: Single Step Protocol for Purification and Thermal Stabilization. Process Biochem. 2006, 41, 2282–2288.
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
INFLUENCE OF pH AND TEMPERATURE ON THE ACTIVITY OF SnO2-BOUND αAMYLASE: A GENOTOXICITY ASSESSMENT OF SnO2 NANOPARTICLES Mohd Jahir Khan
a b
& Qayyum Husain
a c
a
Department of Biochemistry , Faculty of Life Sciences, Aligarh Muslim University , Aligarh , India b
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School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor Darul Ehsan , Malaysia c
College of Medical Sciences, Jazan University , Jazan, Kingdom of Saudi Arabia Accepted author version posted online: 02 Sep 2013.Published online: 05 Feb 2014.
To cite this article: Mohd Jahir Khan & Qayyum Husain (2014) INFLUENCE OF pH AND TEMPERATURE ON THE ACTIVITY OF SnO2-BOUND α-AMYLASE: A GENOTOXICITY ASSESSMENT OF SnO2 NANOPARTICLES, Preparative Biochemistry and Biotechnology, 44:6, 558-571, DOI: 10.1080/10826068.2013.835732 To link to this article: http://dx.doi.org/10.1080/10826068.2013.835732
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Preparative Biochemistry & Biotechnology, 44:558–571, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.835732
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INFLUENCE OF pH AND TEMPERATURE ON THE ACTIVITY OF SnO2-BOUND a-AMYLASE: A GENOTOXICITY ASSESSMENT OF SnO2 NANOPARTICLES
Mohd Jahir Khan1,2 and Qayyum Husain1,3 1 Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India 2 School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor Darul Ehsan, Malaysia 3 College of Medical Sciences, Jazan University, Jazan, Kingdom of Saudi Arabia
& Immobilization of biologically important molecules on a myriad of nanosized materials has attracted great attention due to their small size, biocompatibility, higher surface-to-volume ratio, and lower toxicity. These properties make nanoparticles (NPs) a superior matrix over bulk material for the immobilization of enzymes and proteins. In the present study, Bacillus amyloliquefaciens a-amylase was immobilized on SnO2 nanoparticles by a simple adsorption mechanism. Nanoparticle-adsorbed enzyme retained 90 % of the original enzyme activity. Thermal stability of nanosupport was investigated by thermogravimetric and differential thermal analysis. Scanning electron microscopic studies showed that NPs have porous structure for the high-yield immobilization of a-amylase. The genotoxicity of SnO2-NPs was analyzed by pUC19 plasmid nicking and comet assay and revealed that no remarkable DNA damage occurred in lymphocytes. The pH-optima was found to be the same for both free and SnO2-NPs bound enzyme, while the temperature-optimum for NPs-adsorbed a-amylase was 5 C higher than its free counterpart. Immobilized enzyme retained more than 70% enzyme activity even after its eight repeated uses. Keywords a-amylase, genotoxicity, immobilization, SnO2-NPs, starch hydrolysis
INTRODUCTION Amylases are industrially important enzymes, catalyzing the hydrolysis of starch, glycogen, and other polysaccharides into small molecules.[1] These enzymes are applied in the production of bioethanol and of high fructose and maltose syrups, as well as synthesis of many pharmaceuticals.[2,3] Since the opening of new avenues in the field of biotechnology, Address correspondence to Qayyum Husain, College of Medical Sciences, Jazan University, Jazan, Post Box-114, Kingdom of Saudi Arabia. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/lpbb.
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the spectrum of amylase applications has been expanded into several fields, like clinical, medicinal, and analytical chemistry.[4] Amylases are ubiquitous in nature, are produced by bacteria, yeast, fungi, higher plants, and animals, and play a dominant role in carbohydrate metabolism. In spite of their wide distribution, bacterial amylases are preferred for industrial purposes over the others due to cost-effectiveness, easy production, and simple processing, modification, and optimization.[5] Immobilization of enzyme is important not only for its reusability but also for effective industrial uses. Immobilized enzymes are more robust and resistant to environmental changes as compared to free enzymes.[6] Hence, different immobilization techniques and supports have been investigated and utilized for several years to enhance activity, lifetime, and stability of the commercially useful enzymes like amylases.[7–11] Nanotechnology is now being applied in several fields including biotechnology, physics, chemistry, optics, and mechanics, and it is considered a trigger of the next industrial revolution by many contemporary experts.[12,13] The interaction of protein with the planar surfaces of bulk materials often induces considerable changes in their secondary and tertiary structure, while the high curvature of nanoparticles (NPs) protects their native structure.[14,15] Furthermore, metal NPs have a higher isoelectric point that is suitable for the absorption of proteins with lower isoelectric point, as immobilization is primarily driven by electrostatic interaction.[16] These properties make nanomaterials superior supports as compared to the traditional bulk materials for the immobilization of enzyme and other biomolecules.[17,18] In this study, the immobilization of a-amylase on SnO2-NPs is evaluated simply by adsorption. The surface morphology of NPs before and after binding with enzyme is characterized by scanning electron microscopy (SEM). Thermal stability of SnO2-NPs are studied with thermogravimetric analysis (TGA) and differential thermal analysis (DTA). Genotoxicity of NPs is examined by pUC19 plasmid nicking and comet assay. Immobilized a-amylase is also explored for its applicability and efficiency, including recyclability and stability.
MATERIALS AND METHODS a-Amylase (Bacillus amyloliquefaciens; EC 3.2.1.1) was purchased from Sigma (St. Louis, MO). Starch, maltose, glucose, and 3,5-dinitro-salicylic acid (DNS) were obtained from SRL Chemicals (Mumbai, India). All other chemicals and reagents were of analytical grade and used without further purification.
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Synthesis and Characterization of Nanoparticles
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SnO2-NPs were synthesized and characterized according to the procedure described in our previous study.[19] In short, a sol solution was prepared by dissolving 3.0 g of SnCl4 5H2O in 100 mL methanol under vigorous stirring. Aqueous ammonia solution (4.0 mL) was added drop wise to this solution. The resulting gel was filtered and washed with methanol to remove impurities and dried over 80 C for 5 hr in order to remove water. Finally, dried gel powder was calcined at 400 C for 2 hr, which resulted in formation of SnO2-NPs.
Adsorption of a-amylase on SnO2-NPs SnO2-NPs (100 mg) were added into a-amylase (1820 U) containing 50 mM phosphate buffer, pH 6.0. The immobilization was carried out overnight in a shaking water bath at 25 C. After immobilization, enzyme-bound NPs were collected by centrifugation at 5,000 g for 20 min at 4 C and washed thrice with the same buffer to remove unbound enzyme. Finally, immobilized enzyme was stored in assay buffer at 4 C for further study.
Assay of a-amylase The activity of a-amylase was measured spectrophotometrically by determining the amount of reducing groups formed after enzymatic hydrolysis.[20] Assay solutions contained an appropriate amount of free= immobilized enzyme in starch solution (1.0%, w=v). The reaction mixture was incubated at 50 C for 10 min in a shaking water bath. Reaction was stopped by adding 1.0 mL of DNS reagent and tubes were kept in boiling water for 5 min to develop color. The reaction mixture was diluted with distilled water, and absorbance was recorded by double-beam spectrophotometer at 540 nm. One unit of a-amylase activity is defined as the amount of enzyme that catalyzes the production 1.0 mmol of reducing groups per minute under the standard assay conditions.
Scanning Electron Microscopy The surface characteristics of SnO2-NPs and enzyme-immobilized SnO2-NPs were studied with SEM (Zeiss EVO 50, Germany). To avoid any surface charging, NPs were coated with the film of Au-Pd using a sputter coater. The specimens were examined at 20 kV accelerating voltage.
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TGA and DTA of SnO2-NPs TGA and DTA were applied to investigate the thermal properties and stability of nanosupport in N2. The experiment was performed at a heating rate of 10 C=min with a Mettler-3000 thermal analyzer and TA-Q2000 DSC for TGA and DTA, respectively.
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Genotoxicity Analysis of SnO2-NPs by Plasmid Nicking Assay Conversion of supercoiled plasmid DNA into open circular and further linear form has been used as an index of DNA breakage. A single-strand breaks in DNA can be determined by electrophoresis via differential mobility of supercoiled, open circle, and linear forms of the plasmid. The assay was performed with pUC19 plasmid DNA as described by Kitts et al with minor modifications.[21] Reactions were carried out using 1.0 mg of pUC19 DNA with 50 and 100 mg=mL of NPs in 20 mM Tris-HCl, pH 7.5, at room temperature for 2 hr prior to addition of 10 mL gel loading buffer. The control and treated DNA solutions were loaded on agarose gel and electrophoresis was performed in TBE running buffer. Finally, gel was visualized on an ultraviolet (UV) transilluminator and imaged with a gel documentation system. Alkaline Comet Assay of SnO2-NPs Comet assay (single-cell gel electrophoresis) was performed to examine the genotoxicity of NPs. The result is based on the migration of nuclear DNA to form a tail-like structure. Assay was performed according to the procedure of Dhawan et al. with some modifications.[22] Microscopic slides were coated with 1.0% normal-melting-point (NMP) agarose and allowed to air dry. A mixture of NPs-treated cells (lymphocytes) with 80 mL of 1.0% low-melting-point (LMP) agarose was added to the slides, which were immediately covered with cover slips and kept for 10 min in a refrigerator to solidify. After solidification, cover slips were removed gently and slides were immersed in cold lysing solution (2.5 mM NaCl, 1% Triton X-100, 10% dimethyl sulfoxide [DMSO], 10 mM ethylenediamine tetraacetic acid [EDTA], 10% sodium lauroyl sarcosinate, and 100 mM Tris, pH 10) for 1 hr at 4 C. DNA was kept for 30 min in freshly prepared alkaline electrophoresis solution (30 mM NaOH and 1.0 mM EDTA, pH > 13). Electrophoresis was performed at 4 C in 300 mA current. Afterward, slides were gently immersed in neutralization buffer (0.4 M Tris-HCl, pH 7.5) for 10 min, before a final wash with deionized water and covering with cover slips. Slides were scored with image analysis system (Comet 5.5; Kinetic Imaging, Liverpool, UK) attached to an Olympus fluorescent microscope
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(CX 41, Olympus Optical Randomly selected images DNA damage. Migration micrometer by a Comet 5.5
Co., Tokyo, Japan) at 200 magnification. were analyzed from each sample to assess of DNA from nucleus was measured in image analysis system.
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Influences of pH and Temperature on the Activity of a-amylase The pH optima for free and immobilized enzyme were evaluated by determining the activity of enzyme in buffers of different pH (4.0–8.0) values using different buffers. Molarity of each buffer was 50 mM and the percent maximum activity at pH 6.0 was taken as control (100%) for the calculation of remaining percent activity for soluble and SnO2-NPs bound a-amylase. The temperature optima of soluble and immobilized a-amylase were determined at varying temperatures over the range 20–80 C in a standard buffer (50 mM, pH 6.0). The enzyme activity at 50 C and that at 55 C were taken as control (100%) for calculation of residual percent activity of soluble and immobilized a-amylase, respectively. Reusability of SnO2-NPs Bound a-amylase Reusability of SnO2-NPs bound a-amylase was examined at optimum pH and temperature. After each run, enzyme-bound NPs were separated, washed with assay buffer, then reintroduced into fresh reaction medium. This procedure was repeated for 8 successive days and the activity determined on the first day was considered as control (100%) for calculating residual activity after each repeated use. Estimation of Protein Protein concentration was determined by the protein–dye binding method.[23] Bovine serum albumin was used as the standard protein. RESULTS AND DISCUSSION Immobilization Efficiency of SnO2-NPs-Bound a-amylase Nanoparticles exhibited interesting properties, such as large surface area, high catalytic efficiency, and strong adsorption ability, that make them potential candidates for enzyme immobilization. Nanomaterials strongly bind to several molecules, such as chemical compounds, drugs, probes, and proteins, either covalently or by adsorption.[24,25] Table 1 shows the immobilization efficiency of Bacillus amyloliquifaciens a-amylase on
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a-Amylase Immobilized on SnO2-NPs Activity Bound to SnO2-NPs (U)
Enzyme Loaded (X) (U)
Enzyme Activity in Washes (Y) (U)
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1820
1132
Theoretical (X – Y ¼ A)
Actual (B)
Effectiveness Factor (B=A) ¼ (g)
Activity Yield % (B=A 100)
621
0.90
90%
688
SnO2-NPs by physical adsorption. NPs-adsorbed enzyme retained 90% enzyme activity, which is significantly higher than the earlier reported work. The preserved activity of a-amylase was 86% and 26–47% when covalently immobilized onto poly(2-hydroxyethyl methacrylate) and polyaniline supports, respectively.[26,27] Furthermore, 87% activity yield has been reported for Penicillium griseofulvum a-amylase adsorbed onto Celite.[28] SEM Characterization The binding of a-amylase with SnO2-NPs was investigated by SEM analysis. The result showed that NPs have fine and uniform structure with some agglomerates to form bigger clusters (Figure 1a). After immobilization, surfaces of NPs become rough and covered with enzymes (Figure 1b). SEM images demonstrate that the fine particles were the major population, providing large surface area and porous structure for loading the significant amount of enzyme. TGA and DTA Figure 2 exhibits the thermostability of SnO2-NPs by TGA and DTA. The TGA curve demonstrates that the weight loss at 125 C was 2.7%, which is attributed to removal of water=solvent mixture from the sample (Figure 2a). Additional weight loss (3.4%) was observed at 600 C representing the removal of solvent molecules that were entrapped in the porous SnO2 matrix. DTA study showed that thermal decomposition of the SnO2-NPs begins at a temperature higher than 300 C in air under atmospheric pressure (Figure 2b). Genotoxicity Assessment of SnO2-NPs Genotoxicity is considered one of the most important toxic endpoints in most chemical toxicity testing and risk assessment. The toxicity evaluation of SnO2-NPs is very important if we are going to use them in the food
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FIGURE 1 Scanning electron microscopy for free and enzyme bound SnO2-NPs. Free and aamylase-bound SnO2-NPs were coated with Au-Pd to minimize surface charging. The specimens were observed at 20 kV accelerating voltage and 40k magnification. SEM micrographs show SnO2-NPs (a) and enzyme-bound SnO2-NPs (b).
and pharmaceutical industry. In this study, the genotoxicity of SnO2-NPs was investigated by plasmid nicking and comet assay. Plasmid nicking assay representing the breakage in plasmid DNA was negligible at 100 mg=mL of NPs. The band intensities of the treated samples were almost similar to the control sample. This confirms that SnO2-NPs are not toxic up to 100 mg=mL concentrations (Figure 3). The genotoxicity was further analyzed by comet assay where the extent of DNA migration is directly related to its degradation in lymphocytes.[29] The result showed that the DNA breaking was insignificant up to
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FIGURE 2 Thermogravimetric and differential thermal analysis of nanosupport. TGA (a) was performed with a Mettler 3000 thermal analyzer using 2 mg sample with heating rate of 10 C=min in N2 atmosphere. DTA (b) was also carried in similar heating range by using TA-Q2000, DSC.
100 mg=mL of NPs (Figure 4A). In Figure 4B, the histogram represents the percentage DNA in tail of the comet since tail length is an index of DNA damage. The result showed that no remarkable changes in the tail length were observed at different concentrations of SNO2-NPs. It has been reported that the genotoxicity of NPs is either from direct interaction of NPs with DNA or from release of toxic ions from soluble NPs. Furthermore, generation of oxidative stress is also an important factor that may cause the degradation of DNA in lymphocytes.[30] In a similar study, Jin et al. reported that silica nanocomposites have no significant toxic effects at the molecular and cellular levels below a concentration of 0.1 mg=ml.[31]
Optimum pH and Temperature for the Free and NPs-Bound a-Amylase Stability of enzymes against denaturing agents is an important factor for industrial exploitation. The influence of pH on the activity of soluble and SnO2-NPs adsorbed a-amylase was studied over a range of pH, 4.0–8.0 (Figure 5). The optimum pH for starch hydrolysis was pH 6.0 for both free and adsorbed enzymes. However, the immobilized a-amylase exhibited
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FIGURE 3 Plasmid nicking assay of SnO2-NPs. Toxicity of SnO2-NPs was analyzed by plasmid nicking assay as described in the text. Circular double-stranded pUC19 DNA was used in this study. Lane (C), control pUC19 DNA; (T1) pUC19 DNA treated with 50 mg=mL SnO2-NPs; (T2) pUC19 DNA treated with 100 mg=mL SnO2-NPs.
significant broadening in pH-activity profile as compared to its soluble counterpart. Immobilized a-amylase retained 61% activity at pH 8.0, while soluble enzyme exhibited only 40% activity under similar experimental conditions. The observed higher stability of immobilized a-amylase against pH might be due to binding of enzyme with nanosupport, which prevents the unfolding=denaturation of enzyme.[32] It was presumed that the configuration of a-amylase was fixed on the surface of SnO2-NPs, which may increase the enzyme’s tolerability of pH variance in the surrounding microenvironment. Figure 6 shows the effect of temperature on the activity of soluble and immobilized a-amylase. Soluble enzyme has an optimum temperature of 50 C, whereas the maximum activity of immobilized a-amylase was found at 55 C. Initially, the catalytic activity of free enzyme was enhanced with increasing temperature up to the optimum temperature, followed by a
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FIGURE 4 (A) Comet assay of SnO2-NPs. Quantitative analysis of nuclear DNA damage was observed by Comet assay as given in the text. The microscopic images have been shown for control DNA (C), DNA treated with 50 mg=mL SnO2-NPs (T1), and 100 mg=mL SnO2-NPs (T2). (B) Comet assay histogram of SnO2-NPs. Histogram shows control DNA (C) treated with 50 mg=mL SnO2-NPs (T1) and with 100 mg=mL SnO2-NPs (T2).
FIGURE 5 pH–activity profiles for soluble and immobilized a-amylase. The activity of soluble and immobilized a-amylase was measured in buffers (50 mM) of various pH (pH 4.0–8.0). The molarity of each buffer was 50 mM. The activity at pH 6.0 was taken as control (100%) for calculating remaining percent activity for both enzyme preparations at different pH.
rapid decline in the activity of free enzyme as compared to the immobilized a-amylase. This might be because of adsorption of the enzyme on the support restricting its free movement, providing extended stability even at higher temperatures. Therefore, the enzyme might have higher activation
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FIGURE 6 Temperature–activity profiles for soluble and immobilized a-amylase. The activity for soluble and immobilized a-amylase was assayed at various temperatures (40–80 C). The enzyme activity at 50 C and 55 C was taken as control for calculating remaining percent activity for soluble and NPs bound aamylase, respectively.
FIGURE 7 Reusability of immobilized a-amylase. The reusability of SnO2-NPs bound a-amylase was monitored for 8 successive days. The activity determined on the first day was taken as control (100%) to calculate remaining percent activity after each repeated use.
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energy to show its catalytic activity. Similar results were shown by earlier investigators.[33,34]
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Reusability One of the major hurdles for cost effectiveness of enzymatic processes is the inability of reuse the enzyme in a particular reaction. The immobilized a-amylase can be used repeatedly for starch hydrolysis. It was found that immobilized enzyme was active, holding over 90% activity after 4 repeated cycles and 77% activity even after 8 cycles of use (Figure 7). Thus, the current study agrees with the fact that entailing structural stabilization of immobilized enzyme minimizes the possibility of leaching of enzyme from the support and shows better activity and efficacy.
CONCLUSIONS SnO2-NPs structure provided a favorable microenvironment for the immobilization of a-amylase. The SnO2-NPs-bound enzyme not only broadened its pH optimum but also showed an elevated temperature optimum, overcoming the problems related to the gelatinization of starch during hydrolysis with significantly decreased possible microbial contamination. The genotoxicity study further supports that SnO2-NPs-bound enzyme can be safely used in food industries as well as in health sector. Thus, the system containing SnO2-NPs-adsorbed a-amylase can be exploited for the hydrolysis of starch in batch as well as in continuous systems.
ACKNOWLEDGMENTS The authors gratefully acknowledge EM Central Facility, Textile Technology Department, IIT Delhi, India, for SEM analysis. The authors are also thankful to the University Grants Commission, New Delhi, India, for sponsoring a fellowship to one of us (M. J. Khan).
REFERENCES 1. Ben Abdelmalek-Khedher, I.; Urdaci, M.C.; Limam, F.; Schmitter, J.M.; Marzouki, M.N.; Bressollier, P. Purification, Characterization and Partial Primary Sequence of a Major-Maltotriose-Producing a-Amylase, ScAmy43, From Sclerotinia sclerotiorum. J. Microb. Biotechnol. 2008, 18, 1555–1563. 2. Sharma, A.; Satyanarayana, T. Optimization of Medium Components and Cultural Variables for Enhanced Production of Acidic High Maltose-Forming and Ca2þ-Independent a-Amylase by Bacillus acidicola. J. Biosci. Bioeng. 2011, 111, 550–553.
Downloaded by [Florida International University] at 21:21 05 January 2015
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3. Sivaramakrishnan, S.; Gangadharan, D.; Nampoothiri, K.M.; Soccol, C.R.; Pandey, A. a-Amylases From Microbial Sources: An Overview on Recent Developments. Food Technol. Biotechnol. 2006, 44, 173–184. 4. Gupta, R.; Gigras, P.; Mohapatra, H.; Goswami, V.K.; Chauhan, B. Microbial a-Amylases: A Biotechnological Perspective. Process Biochem. 2003, 38, 1599–1616. 5. Pandey, A.; Nigam, P.; Soccol, C.R.; Soccol, V.T.; Singh, D.; Mohan, R. Advances in Microbial Amylases. Biotechnol. Appl. Biochem. 2000, 31, 135–152. 6. Khan, M.J.; Husain, Q.; Ansari, S.A. Polyaniline-Assisted Silver Nanoparticles: A Novel Support for the Immobilization of a-Amylase. Appl. Microbiol. Biotechnol. 2013, 97, 1513–1522. 7. Kahraman, M.V.; Bayramoglu, G.; Kayaman-Apohan, N.; Gungor, A. a-Amylase Immobilization on Functionalized Glass Beads by Covalent Attachment. Food Chem. 2007, 104, 1385–1392. 8. Tee, B.L.; Kaletunc, G. Immobilization of a Thermostable a-Amylase by Covalent Binding to an Alginate Matrix Increases High Temperature Usability. Biotechnol. Prog. 2009, 25, 436–445. 9. Cordeiro, A.L.; Lenk, T.; Werner, C. Immobilization of Bacillus licheniformis a-Amylase Onto Reactive Polymer Films. J. Biotechnol. 2011, 154, 216–221. 10. Yang, K.; Xu, N.S.; Su, W.W. Co-Immobilized Enzymes in Magnetic Chitosan Beads for Improved Hydrolysis of Macromolecular Substrates Under a Time-Varying Magnetic Field. J. Biotechnol. 2010, 148, 119–127. 11. Sun, Y.M.; Chen, J.P.; Chu, D.H. Preparation and Characterization of a-Amylase Immobilized Thermal Responsive Composite Hydrogel Membranes. J. Biomed. Mater. Res. 1999, 45, 125–132. 12. Salata, O.V. Applications of Nanoparticles in Biology and Medicine. J. Nanobiotechnol. 2004, 2, 1–6. 13. Ansari, S.A.; Husain, Q. Potential Applications of Enzymes Immobilized on=in Nano Materials: A Review. Biotechnol. Adv. 2012, 30, 512–523. 14. Li, J.; Wang, J.; Gavalas, V.G.; Atwood, D.A.; Bachas, L.G. Alumina-Pepsin Hybrid Nanoparticles With Orientation-Specific Enzyme Coupling. Nano Lett. 2003, 3, 55–58. 15. Chow, D.C.; Johannes, M.S.; Lee, W.-K.; Clark, R.L.; Zauscher, S.; Chilkoti, A. Nanofabrication With Biomolecules. Mater. Today 2005, 8, 30–39. 16. Grealis, C.; Magner, E. The Oxidation of Cytochrome c in Nonaqueous Solvents. Chem. Commun. 2002, 8, 816–817. 17. Topoglidis, E.; Campbell, C.J.; Cass, A.E.G.; Durrant, J.R. Factors That Affect Protein Adsorption on Nanostructured Titania Films. A Novel Spectroelectrochemical Application to Sensing. Langmuir 2001, 17, 7899–7906. 18. Khan, M.J.; Husain, Q.; Azam, A. Immobilization of Porcine Pancreatic a-Amylase on Magnetic Fe2O3 Nanoparticles: Applications to the Hydrolysis of Starch. Biotechnol. Bioprocess. Eng. 2012, 17, 377–384. 19. Khan, M.J.; Qayyum, S.; Alam, F.; Husain, Q. Effect of Tin Oxide Nanoparticle Binding on the Structure and Activity of a-Amylase From Bacillus amyloliquefaciens. Nanotechnology 2011, 22, 455708– 455715. 20. Bernfeld, P. Amylase, a and b. In Methods in Enzymology. Colowick, S.P., Kaplan, N.O., Eds.; Academic Press, New York, NY, 1955; p. 149. 21. Kitts, D.D.; Wijewickreme, A.N.; Hu, C. Antioxidant Properties of a North American Ginseng Extract. Mol. Cell. Biochem. 2000, 203, 1–10. 22. Dhawan, A.; Bajpayee, M.; Parmar, D. Comet Assay: A Reliable Tool for the Assessment of DNA Damage in Different Models. Cell. Biol. Toxicol. 2009, 25, 5–32. 23. Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein–Dye Binding. Anal. Biochem. 1976, 72, 248–254. 24. Chen, B.; Pernodet, N.; Rafailovich, M.H.; Bakhtina, A.; Gross, R.A. Protein Immobilization on Epoxy-Activated Thin Polymer Films: Effect of Surface Wettability and Enzyme Loading. Langmuir 2008, 24, 13457–13464. 25. Luo, X.L.; Xu, J.J.; Du, Y.; Chen, H.Y. A Glucose Biosensor Based on Chitosan-Glucose Oxidase-Gold Nanoparticles Biocomposite Formed by One-Step Electrodeposition. Anal. Biochem. 2004, 334, 284–289. 26. Arica, M.Y.; Hasirci, V.; Alaeddinoglu, N.G. Covalent Immobilization of a-Amylase Onto pHEMA Microspheres: Preparation and Application to Fixed Bed Reactor. Biomaterials 1995, 16, 761–768. 27. Ashly, P.C.; Joseph, M.J.; Mohanan, P.V. Activity of Diastase a-Amylase Immobilized on Polyanilines (PANIs). Food Chem. 2011, 127, 1808–1813.
Downloaded by [Florida International University] at 21:21 05 January 2015
Genotoxicity Assessment of SnO2 Nanoparticles
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28. Ertan, F.; Yagar, H.; Balkan, B. Some Properties of Free and Immobilized a-Amylase From Penicillium griseofulvum by Solid State Fermentation. Prep. Biochem. Biotechnol. 2006, 36, 81–91. 29. Collins, A.R.; Oscoz, A.A.; Brunborg, G.; Gaivao, I.; Giovannelli, L.; Kruszewski, M.; Smith, C.C.; Stetina, R. The Comet Assay: Topical Issues. Mutagenesis 2008, 23, 143–151. 30. Moreira, S.; Silva, N.B.; Almeida-Lima, J.; Rocha, H.A.; Medeiros, S.R.; Alves, C., Jr.; Gamma, F.M. BC Nanofibres: In Vitro Study of Genotoxicity and Cell Proliferation. Toxicol. Lett. 2009, 189, 235–241. 31. Jin, Y.; Kannan, S.; Wu, M.; Zhao, J.X. Toxicity of Luminescent Silica Nanoparticles to Living Cells. Chem. Res. Toxicol. 2007, 20, 1126–1133. 32. Varavinit, S.; Chaokasem, N.; Shobsngo, S. Immobilization of a Thermostable a-Amylase. Sci. Asia 2002, 28, 247–251. 33. Ravian, P.; Tang, J.; Rasco, B.A. Thermal Stability of a-Amylase From Aspergillus oryzae Entrapped in Polyacrylamide Gel. J. Agric. Food Chem. 2003, 51, 5462–5466. 34. Kumar, R.S.S.; Vishwanath, K.S.; Singh, S.A.; Rao, A.G.A. Entrapment of a-Amylase in Alginate Beads: Single Step Protocol for Purification and Thermal Stabilization. Process Biochem. 2006, 41, 2282–2288.
