List of publications and presentations

Publications and presentations

158

LIST OF PUBLICATIONS AND PRESENTATIONS

PUBLICATIONS:

1. Mohammad Jahir Khan, Shariq Qayyum, Fahad Alam and Qayyum Husain.

Effect of tin oxide nanoparticle binding on the structure and activity of

α-amylase from Bacillus amyloliquefaciens. Nanotechnology (2011) 22:

455708-455715.

2. Mohammad Jahir Khan, Qayyum Husain and Ameer Azam. Immobilization

of porcine pancreatic α-amylase on magnetic Fe2O3 nanoparticles:

Applications to the hydrolysis of starch. Biotechnology and Bioprocess

Engineering. (2012) DOI 10.1007/s12257-011-0105-8.

3. Mohammad Jahir Khan, Fahim Halim Khan and Qayyum Husain.

Application of immobilized Ipomoea batata β amylase in the saccharification

of starch. Journal of Applied Biological Sciences. (2011) 5: 33-39.

4. Mohammad Jahir Khan, Qayyum Husain and Sajid Ali Ansari. Polyaniline

assisted silver nanoparticles: a novel support for immobilization of α-amylase.

Journal of Chemical Technology and Biotechnology. (Communicated).

5. Mohammad Jahir Khan and Qayyum Husain. High yield immobilization

and stabilization of Bacillus amyloliquifaciens α-amylase by SnO2

nanoparticles. Journal of Nanoscience and Nanotechnology. (Communicated).

6. Mohammad Jahir Khan and Qayyum Husain. Potential applications of

soluble and immobilized amylases: A review. (Communicated).

PRESENTATIONS:

1. Mohammad Jahir Khan, Shariq Qayyum, Fahad Alam and Qayyum Husain.

“Effect of tin oxide nanoparticle binding on the structure and activity of α-

amylase from Bacillus amyloliquefaciens”. Poster presented in Silver Jubilee

Symposium under the auspices of UGC-DRS-II Programme organized by

Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh, India,

March 6, 2012.

2. Mohammad Jahir Khan, Qayyum Husain and Ameer Azam.

“Immobilization of porcine pancreatic α-amylase on magnetic Fe2O3

Publications and presentations

159

nanoparticles: Applications to the hydrolysis of starch”. Poster presented in

International Conference on Chemistry: Frontiers and Challenges; organized

by Department of Chemistry, Faculty of Science, AMU, Aligarh, India, March

5-6, 2011.

3. Mohammad Jahir Khan, Fahim Halim Khan and Qayyum Husain.

“Application of immobilized Ipomoea batata β amylase in the saccharification

of starch”. Poster presented in National Symposium 2011 on “Current Trends

in Biochemical, Biomedical and Environmental Sciences” under the UGC-

DRS II Programme, organized by Department Biochemistry, Faculty of Life

Sciences, AMU, Aligarh, India, February 22, 2011.

4. Pragya Tiwari, Mohammad Jahir Khan, NS Sangwan and RS Sangwan.

“Insilico approach for mining of glucosyl transferase gene sequences for

cloning and functional characterization in Centella asiatica”. Poster presented

in National Interactive Meet, 2008 organized by CIMAP, Lucknow, India,

November 29-30, 2008.

5. Participated in SFFR Satellite India Conference organized by Department of

Biochemistry, AIIMS, New Delhi, India, February 11-12, 2008.

6. Participated in a workshop entitled NMR Spectroscopy and its applications

organized by Indian Society of Analytical Scientists (ISAS)-Delhi Chapter and

Department of Chemistry, Jamia Hamdard, New Delhi, India, August 18-19,

2006.

Effect of tin oxide nanoparticle binding on the structure and activity of α-amylase from Bacillus

amyloliquefaciens

This article has been downloaded from IOPscience. Please scroll down to see the full text article.

2011 Nanotechnology 22 455708

(http://iopscience.iop.org/0957-4484/22/45/455708)

Download details:

IP Address: 14.139.43.130

The article was downloaded on 24/03/2012 at 03:47

Please note that terms and conditions apply.

View the table of contents for this issue, or go to the journal homepage for more

Home Search Collections Journals About Contact us My IOPscience

IOP PUBLISHING NANOTECHNOLOGY

Nanotechnology 22 (2011) 455708 (7pp) doi:10.1088/0957-4484/22/45/455708

Effect of tin oxide nanoparticle binding onthe structure and activity of α-amylasefrom Bacillus amyloliquefaciens

Mohammad Jahir Khan1, Shariq Qayyum2, Fahad Alam3 andQayyum Husain1,4

1 Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University,Aligarh-202002, India2 Department of Agricultural Microbiology, Faculty of Agricultural Sciences,Aligarh Muslim University, Aligarh-202002, India3 Centre of Excellence in Material Science (Nanomaterials), Z H College of Engineering&Technology, Aligarh Muslim University, Aligarh-202002, India

E-mail: [email protected]

Received 8 June 2011, in final form 27 September 2011Published 21 October 2011Online at stacks.iop.org/Nano/22/455708

AbstractProteins adsorbed on nanoparticles (NPs) are being used in biotechnology, biosensors and drugdelivery. However, understanding the effect of NPs on the structure of proteins is still in anascent state. In the present paper tin oxide (SnO2) NPs were synthesized by the reaction ofSnCl4·5H2O in methanol via the sol–gel method and characterized by x-ray diffraction (XRD),Fourier transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM).The binding of these SnO2–NPs with α-amylase was investigated by using UV–vis,fluorescence and circular dichroism (CD) spectroscopic techniques. A strong quenching oftryptophan fluorescence intensity in α-amylase was observed due to formation of a ground statecomplex with SnO2–NPs. Far-UV CD spectra showed that the secondary structure ofα-amylase was changed in the presence of NPs. The Michaelis–Menten constant (Km), wasfound to be 26.96 and 28.45 mg ml−1, while Vmax was 4.173 and 3.116 mg ml−1 min−1 for freeand NP-bound enzyme, respectively.

1. Introduction

α-Amylases (alternative names 1,4-α-D-glucan glucanohydro-lase, glycogenase, E.C.3.2.1.1) are endo-acting enzymes whichbelong to the glycoside hydrolase family 13 (GH13) [1]. Theseenzymes catalyze random hydrolysis of α-1,4-glycosidicbonds in amylose, amylopectin and related polysaccharidesto low molecular weight products such as dextrins, maltoseand glucose molecules [2, 3]. α-Amylases are very importantcommercial enzymes with a number of applications includingtheir use in food, paper, detergent, pharmaceutical and textileindustries [4]. Although α-amylases can be derived fromvarious sources, their industrial demand has been satisfied bymicrobial and recombinant enzymes. The most abundantly

4 Address for Correspondence: Faculty of Applied Medical Science, JazanUniversity, Jazan, Post Box-2092, Kingdom of Saudi Arabia.

used bacterial α-amylases for industrial purposes are derivedfrom Bacillus species [5]. These enzymes have beencharacterized from a wide variety of organisms and almost allof them have a similar structure and catalytic mechanism [6, 7].

The interaction of enzymes with ligands offered stim-ulating opportunities for a wide variety of applications inthe field of biotechnology and medicine [8–10]. Generallyproteins undergo structural changes when interacting withligands. Metal oxide NPs serves as novel candidates forthe binding of protein due to their large surface-to-volumeratio, high biocompatibility, non-toxicity, chemical stabilityand ease of preparation [11, 12]. Adsorption of proteins onsolid surfaces strongly depends on the nature of the proteinand surface geometry and physicochemical characteristics ofthe solid surface. Binding of enzymes directly on the nakedsurface of bulk materials may result in their denaturation and

0957-4484/11/455708+07$33.00 © 2011 IOP Publishing Ltd Printed in the UK & the USA1

Nanotechnology 22 (2011) 455708 M J Khan et al

loss of activity. However, enzymes adsorbed onto NP surfacesretained their activity due their biocompatible nature [13, 14].Various specific and nonspecific interactions such as elec-trostatic, hydrogen bonding and hydrophobic interactions areinvolved in the adsorption of protein on NP surfaces whichaffect the structure and stability of proteins [15, 16].

The present study is focused on the interaction of α-amylase with tin oxide NPs. Enzyme adsorbed NPs werecharacterized by x-ray diffraction (XRD), Fourier transforminfrared spectroscopy (FT-IR) and transmission electronmicroscopy (TEM). Structural changes in enzymes afterbinding with NPs were analyzed by UV–vis, fluorescence andcircular dichroism (CD) spectroscopy. Kinetic parameters offree and NP-bound enzyme were also evaluated.

2. Materials and methods

2.1. Materials

α-Amylase (from Bacillus amyloliquefaciens, EC.3.2.1.1) andtin tetrachloride pentahydrate (SnCl4·5H2O) were purchasedfrom Sigma-Aldrich Co. (USA). Starch, maltose, glucoseand 3,5-dinitro-salicylic acid (DNS) were obtained from SRLChemicals (Mumbai, India). All other chemicals and reagentswere of analytical grade and used without further purification.

All the experiments were performed in 50 mM sodiumphosphate buffer, pH 6.0 except where specified. The con-centration of protein was determined spectrophotometricallyusing E1%

280 nm = 2.493 on a Shimadzu UV-1700 UV–visSpectrophotometer and alternatively by a protein–dye bindingmethod [17].

2.2. Synthesis of nanoparticles

Tin oxide NPs were synthesized by the sol–gel methodaccording the procedure described Adnan et al with somemodifications [18]. The sol solution was prepared bydissolving 3.0 g of SnCl4·5H2O in 100 ml methanol (CH3OH)under vigorous stirring. Aqueous ammonia solution (4.0 ml)was added drop wise to the above solution under similarexperimental conditions. The resulting gel was filtered andwashed with methanol to remove impurities and dried over80 ◦C for 5 h in order to remove water. Finally, the driedgel powder was calcined at 400 ◦C for 2 h which resulted intoformation of SnO2-NPs.

2.3. Enzyme–nanoparticle interaction studies

A stock solution of protein (5.0 mM) was prepared in 50 mMsodium phosphate buffer, pH 6.0 and diluted with the samebuffer as necessary. The α-amylase concentration was keptconstant at 24.4 μM, 7.0 μM and 5.0 μM, respectively,for UV–vis, CD and fluorescence measurements, while theconcentration of sol–gel synthesized SnO2-NPs was variedfrom 0.2 to 1.0 mM. The reaction mixture was equilibratedfor the optimal incubation time at 25 ◦C for 1 h. Subsequently,UV–vis absorption, fluorescence and CD spectra of the solutionwere recorded to monitor the interaction between α-amylaseand NPs.

2.4. Physical characterization of NPs

The interactions of enzyme with NPs were investigated by FT-IR analysis on an INTERSPEC 2020 (USA) in the range of400–4000 cm−1. The calibration was done with polystyrenefilm. The samples were injected with a Hamiet 100 μl syringeinto the attenuated total reflectance (ATR) box. The syringewas first washed with acetone followed by distilled water.The morphology and size of the SnO2-NPs with and withoutconjugated α-amylase was observed with a JEOL JEM-2100Ftransmission electron microscope with an accelerating voltageof 200 kV. Samples for TEM analysis were prepared by drop-coating diluted NP solution on carbon-coated copper gridsat normal atmospheric conditions. XRD analysis of NPswas done with a Rigaku Miniflex x-ray diffractometer, usinga monochromatized x-ray beam with nickel-filtered Cu Kα

radiation. The diffraction angle was set at between 20◦ and80◦. The crystalline size was determined from correspondingx-ray spectral peaks by the Debye–Scherrer formula:

D = 0.9λ/B cos θ (1)

where λ is the x-ray wavelength (1.540 60 A), B is thefull width at half-maximum of SnO2(110) line and θ is thediffraction angle.

2.5. UV–vis, fluorescence and CD spectroscopy

Absorbance measurements were carried out with ShimadzuUV-1700 spectrometer operated at a resolution of 2.0 nm(Shimadzu, Kyoto, Japan). Enzyme concentration was keptconstant at 24.4 μM in each reaction mixture while the NPconcentration varied from 0.2 to 1.0 mM. Unless otherwisestated, the UV–vis spectra were recorded in a wavelength rangeof 250–350 nm. For sample measurements, the baseline wasalways set with a relevant blank. The final spectrum is anaverage of three independent measurements.

Fluorescence analyses were performed on a ShimadzuSpectrofluorophotometer (RF-5301) equipped with a DR-3data recorder. The fluorescence spectra were measured at25 ± 0.10 ◦C with a 1.0 cm path length. Both excitation andemission slits were set at 5.0 nm. The intrinsic fluorescenceof a protein sample was measured by exciting at 280 nm andemission spectra were recorded in the range of 300–500 nm.

The CD measurements were carried out on Jasco Spectro-polarimeter (J-720). The instrument was calibrated with d-10-camphorsulfonic acid. All the CD measurements were carriedout at 25 ◦C using a thermostatically controlled cell holderattached to a Neslab RTE-110 water bath with an accuracyof 0.10 ◦C. Far-UV CD spectra were measured at a proteinconcentration of 7.0 μM. The path length of the cell was1.0 mm.

Mean residue ellipticity (MRE) = θobs/(10 ×n × l × cp) (2)

where θobs is the CD in millidegrees, N is the number of aminoacid residues (483), l is the path length of the cell and cp is themole fraction. The % α-helical content was calculated fromthe MRE values at 222 nm using the following equation asdescribed by Chen et al [19].

% α-helix = (MRE222 nm − 2340/30300) × 100. (3)

2


Figure 1. XRD pattern of SnO2-NPs mediated in methanol. Thex-ray diffraction pattern of SnO2-NPs was recorded at roomtemperature using a Rigaku Miniflex x-ray diffractometer withCu Kα radiation (λ = 1.540 60 A) in 2θ ranging from 20◦ to 80◦.

2.6. Assay of α-amylase activity

α-Amylase activity was determined according to the procedureof Bernfeld et al with some modifications [20]. Appropriatelydiluted enzyme was added to 500 μl of gelatinized starch (1%)solution. After incubation at 50 ◦C for 10 min in a shakingwater bath, the reaction was stopped by adding 1.0 ml of DNSreagent. The tubes were kept in boiling water for 5 min todevelop color. After cooling, 1.0 ml distilled water was addedand finally absorbance was recorded at 540 nm.

One unit (1.0 U) of α-amylase activity is defined asthe amount of enzyme that liberates 1.0 mM of maltosemin−1 under standard assay conditions. A standard curveof absorbance against amount of maltose was prepared tocalculate the amount of maltose released during the assay.

2.7. Statistical analysis

Each value represents the mean for three independentexperiments performed in duplicate, with average SD of <5%.Data were analyzed by one-way ANOVA and p-values < 0.05were considered statistically significant.

3. Results and discussion

3.1. Biophysical characterization of free and enzyme boundSnO2-NPs

Among various methods for chemical synthesis of nanoma-terials, the sol–gel process offered several advantages likelower processing temperature, better homogeneity, controlledstoichiometry and flexibility of forming nanoparticles. Metaloxide NPs have attracted much attention owing to theirremarkable physicochemical properties like high surface area,high electrical conductivity, good chemical stability andsignificant mechanical strength, therefore these NPs havebeen widely applied for the immobilization of wide range ofbiomolecules [21–23].

Figure 1 shows XRD patterns of SnO2-NPs calcined at400 ◦C for 2 h using methanol as a solvent. Samples exhibiteda wide diffraction peak at the same position, which can beindexed to the tetragonal structure of SnO2-NPs (JCPDS no.88-0287). The XRD diffractogram showed broad peaks at 2θ

of 27◦, 34◦, and 53◦ which correspond to the diffraction of110, 101 and 211 planes of SnO2 crystal, respectively. Thesize of the nanocrystals was calculated by using Scherrer’sequation (1). Based on the full width at half-maximum of the110 diffraction plane, the average crystallite size of SnO2-NPswas found to be 11.2 nm.

The binding of α-amylase with SnO2-NPs was confirmedby FT-IR analysis. Figure 2 shows the IR spectra of NPs, α-amylase and enzyme-bound NPs. The intense and broad peak

Figure 2. FT-IR spectra of SnO2-NPs, α-amylase and enzyme-bound SnO2-NPs. FT-IR spectra of SnO2-NPs (a), α-amylase (b) andenzyme-bound SnO2-NPs (c) were monitored with an INTERSPEC 2020 (USA). The calibration was done with polystyrene film. The syringewas first washed with acetone followed by distilled water. The samples were injected into the ATR box by a Hamiet 100 μl syringe.

3


Figure 3. TEM images of SnO2-NPs (A) and enzyme-boundSnO2-NPs (B) were recorded with a JEOL JEM-2100F transmissionelectron microscope with an accelerating voltage of 200 kV.

at 3346 cm−1 and 1638 cm−1 (figure 2(a)) can be attributedto the O–H vibration of methanol and water absorbed on thesample surface [24]. The metal oxide exhibited a peak at554 cm−1 which could be assigned due to stretching vibrationof the Sn–O framework (figures 2(a) and (c)). The bandintensity of protein at 1643 cm−1 showed the bending vibrationof the N–H group which decreased (4.0 units) after adsorptionof enzyme on the NP surface (figures 2(b) and (c)). Othercharacteristic bands at 1110 and 1036 cm−1 could be due tothe stretching and bending vibrations of aliphatic amines. Thestrong absorption peak at 1212 cm−1 (figure 2(a)) was due tointeraction of the NH group of α-amylase with the Sn–O groupof NPs [25, 26].

Figure 4. UV–vis absorption spectra of α-amylase in the presence ofSnO2-NPs. A fixed concentration of α-amylase (24.4 μM) wasmixed with varying concentrations of SnO2-NPs and their spectrawere recorded in the UV–visible region.

TEM images of NPs before and after conjugation withenzyme further support the binding of α-amylase with SnO2-NPs. Figure 3(A) shows an electron micrograph of free NPsat a magnification of 50 000×, and it is revealed that nearlyspherical SnO2-NPs with a mean diameter of 25 nm of aredistributed uniformly with some aggregation due overlappingof some bigger particles with the smaller particles. TheNPs remain spherical after adsorption of enzyme but themean diameter increased from 25 to 32 nm (figure 3(B)).This suggests that the conjugation process did not change themorphology of the NPs while the size has increased. Thismight be due to the adsorption of a significant amount ofenzyme on the NP surfaces.

3.2. UV–vis and fluorescence spectra of native and NP boundα-amylase

UV–vis spectroscopy is an effective and simple tool to explorestructural change in protein molecules [27, 28]. The UV–visspectra obtained by the NP titration experiment demonstratethat the protein microenvironment at pH 6.0 was perturbed(figure 4). Upon addition of an increasing concentration ofSnO2-NPs a consecutive regular increase in the absorbancemaxima of protein at 280 nm is shown. This might be dueto the partial or ful adsorption of enzyme on the surface of theNPs. These results indicate that there is an interaction betweenSnO2-NPs and α-amylase through ground state complexformation [29].

The conformational changes of α-amylase in the presenceof SnO2-NPs were evaluated by measurement of the intrinsicfluorescence intensity of tryptophan residues of protein(figure 5). NPs having diameter in the range 30–70 nmefficiently quench α-amylase, as evidenced by the progressivedecrease in the emission maximum intensity. This quenchingeffect indicated that SnO2-NPs strongly interact with thechromophore residues of protein (figure 6). The tendencyof red-shift to occur in the protein upon binding of NPssuggests that the interaction was specific and involved only a

4


Figure 5. Fluorescence spectra of α-amylase were recorded in therange 300–500 nm in the presence of varying concentrations ofSnO2-NPs (from a → e are 0.0, 0.2, 0.4, 0.6 and 1.0 mM,respectively). Inset: change in fluorescence intensity (FI) at 340 nmwith NPs as monitored by intrinsic fluorescence by exciting proteinat 280 nm.

Figure 6. Stern–Volmer plots of NP quenching for α-amylase. Theenzyme concentration was kept constant (5.0 μM) while the NPconcentration increased from 0.0 to 1.0 mM. Values shown are theratios of fluorescence in the absence of NPs (Fo) to the fluorescenceat that concentration of quencher (F).

limited part of the protein [30]. The change in fluorescenceemission maxima suggests the solvent exposure of some ofthe tryptophan residue that was in the hydrophobic coreof the protein under native conditions. Quenching offluorophores by metallic NPs has also been reported by earlierinvestigators [31–33].

3.3. Effect of NP binding on the secondary structure of theenzyme

CD is a powerful analytical tool for studying the interactionof proteins with other molecules and determining the

Figure 7. Far-UV CD spectra of α-amylase at different NPconcentrations. Spectra were recorded in the wavelength region200–250 nm. A constant enzyme concentration 7.0 μM was used ineach set of experiments while the NP concentration was 0.0 to1.0 mM. Inset: change in MRE value of α-amylase at 222 nm withincreasing concentration of NPs. The NP concentration was 0.0 to1.0 mM, while the protein concentration was 7.0 μM.

Table 1. Effect of SnO2-NP binding on the secondary structure ofα-amylase. [Note: the MRE value in deg cm2 dmol−1 is calculatedby equation (2). The % α-helix content was calculated by the Chenet al method (equation (3)) [19]. The % β-sheet content wascalculated by online K2d software [38].]

α-helix β-sheetMRE222 nm (%) (%)

Native α-amylase −12 533 49 21α-amylase +0.2 mM SnO2-NPs −13 625 52 22α-amylase +0.6 mM SnO2-NPs −13 975 53 22α-amylase +1.0 mM SnO2-NPs −15 203 57 19

protein conformation in solution or adsorbed onto othermolecules [34]. Figure 7 shows CD spectra of α-amylaserecorded in the wavelength range of 200 to 250 nm; the resultsare expressed as mean residual ellipticity in millidegrees.The result showed that a relatively more compact structureof protein was obtained in presence of NPs, as shown by amore negative spectrum in far-UV CD (figure 7). The CDspectra displayed characteristic peaks (intensive positive peakat around 190 nm and two negative double humped peaksat 208 and 222 nm) of a high α-helical content in nativeenzyme [35]. In the presence of NPs, an increase in the α-helix was observed (table 1), which suggested the interactionbetween SnO2-NPs and α-amylase. The increased percentageof α-helical protein structure showed that NPs bound to theamino acid residues of the polypeptide chain of α-amylase,thus increasing the hydrogen bonding networks.

3.4. Kinetic studies

Catalytic properties of free and NP-bound enzymes wereevaluated with soluble starch as a substrate. The Michaelis–

5


Figure 8. Lineweaver–Burke plot for free α-amylase (open square)and NP-bound α-amylase (filled square). All the samples wereassayed in 50 mM phosphate buffer, pH 6.0 at 50 ◦C in the presenceof a variable amount of starch. The intercept on the y-axiscorresponds to 1/Vmax and the intercept on the x-axis to −1/Km .Data presented are an average of values ±SD of n = 3 experiments.

Menten constant (Km) and maximum activity (Vmax) weredetermined at pH 6.0 and 50 ◦C (figure 8). The valuesof Km in the present enzymatic assay were found to be26.96 and 28.45 mg ml−1 for the free and immobilized α-amylase, respectively. The smaller value of Km in thecase of free enzyme indicates that free enzyme has a higheraffinity for the substrate. The value of Vmax obtained forthe immobilized α-amylase was 3.116 mg ml−1 min−1 whichis one order of magnitude lower than that of free enzyme(4.173 mg ml−1 min−1). The increasing Km value of NP-bound enzyme is might be due to structural changes of theenzyme in the presence of NPs brought about by a higherrigidity and compactness in protein structure and diffusionlimitations of the substrate [36, 37]. The secondary and tertiarystructure of the enzyme is known to play an important role inthe enzyme activity, as the rearrangement and conformationalchanges in these structures may result in the enhancement orsuppression of enzyme activity.

4. Conclusions

Enzyme dysfunction is related to many diseases. It is desirableto be able to regulate enzyme conformation and function. NPscan be selected to specifically bind enzymes and control theirfunctions after surface modifications, providing a promisingstrategy for therapy. In this study, SnO2-NPs were synthesizedusing a simple sol–gel method and were characterized byXRD, FT-IR and TEM analysis. The interaction betweenα-amylase and NPs was evaluated by UV–vis, fluorescenceand CD spectroscopic studies. The analysis of CD spectraindicated that the α-helical content increases considerably inthe presence of NPs. In comparison with native enzyme,NP-bound enzyme revealed significant improvement in thestability of the α-amylase which could henceforth contributeto its better use in various analytical, diagnostic and clinical

applications. Furthermore, the reactors containing such SnO2-NP adsorbed enzyme could be exploited for the hydrolysis ofstarch in batch systems as well as in continuous systems in thenear future.

Acknowledgments

We are thankful to the University Grant Commission,Government of India for financial support to one of us (MJK).The authors are also thankful to AIRF, JNU, New Delhi, forTEM analysis. Dr Faisal M Al-Tobaigy, Dean, Faculty ofApplied Medical Science, Jazan University, Jazan, Kingdomof Saudi Arabia is gratefully acknowledged for help during thewriting of this manuscript.

References

[1] Henrissat B A 1991 Biochem. J. 280 309[2] Maarel M J E C, Van der Veen B, Uitdehaag J C M,

Leemhuis H and Dijkhuizen L 2002 J. Biotechnol. 94 137[3] Burhan A, Nisa U, Gokhan C, Omer C, Ashabil A and

Osman G 2003 Process. Biochem. 38 1397[4] Haki G D and Rakshit S K 2003 Biores. Technol. 89 17[5] Dey G, Palit S, Banerjee R and Maiti B R 2002 J. Ind. Microb.

Biotechnol. 28 193[6] Nielsen J E and Borchert T V 2000 Biochim. Biophys. Acta

1543 253[7] MacGregor E A, Janecek S and Svensson B 2001 Biochim.

Biophys. Acta 1546 1[8] Nel A, Xia T, Madler L and Li N 2006 Science 311 22[9] Fan H, Yang K, Boye D M, Sigmon T, Malloy K J, Xu H and

Brinker C J 2004 Science 304 567[10] Iseult L and Dawson K A 2008 Nanotoday 3 40[11] Ispas C, Sokolov I and Andreescu S 2009 Anal. Bioanal. Chem.

393 543[12] Mizuki T, Watanabe N, Nagaoka Y, Fukushima T,

Morimoto H, Usami R and Maekawa T 2010 Biochem.Biophys. Res. Commun. 393 779

[13] Cao G 2004 Nanostructures and Nanomaterials: Synthesis,Properties and Applications (UK: Imperial College Press)

[14] Wu Z, Zhang B and Yan B 2009 Int. J. Mol. Sci. 10 4198[15] Lei C, Shin Y, Liu J and Ackerman E J 2002 J. Am. Chem. Soc.

124 11242[16] Pascoal A M, Mitidieri S and Fernandes K F 2010 Food

Bioprod. Process. at press[17] Bradford M M 1976 Anal. Biochem. 72 248[18] Adnan R, Razana N A, Rahman I A and Farrukh M A 2010

J. Chin Chem. Soc. 57 222[19] Chen Y H, Yang J T and Martinez H 1972 Biochemistry

11 4120[20] Bernfield P 1955 Methods in Enzymology vol 1,

ed S P Colowick and N O Kaplan (New York: Academic)[21] Brown K R, Fox A P and Natan M J 1996 J. Am. Chem. Soc.

118 1154[22] Wang J 2005 Analyst 130 421[23] Chopra N, Gavalas V G, Bachas L G, Hinds B J and

Bachas L G 2007 Anal. Lett. 40 2067[24] Neng-Qin J, Juan X, Ming-Hua S and Zhi-Yu J 2005 Anal. Lett.

38 1237[25] Milsom E V, Dash H A, Jenkins T A, Halliwell C M,

Thetford A, Bligh N, Nogala W, Opallo M and Marken F2007 J. Electroanal. Chem. 610 28

[26] Zou G L, Liu R, Chen W X and Xu Z D 2007 Mater. Res. Bull.42 1153

[27] Zhang J and Gao L 2004 J. Solid. State Chem. 177 1425

6


[28] Hu D H, Wu H M, Liang J G and Han H Y 2008 Spectrochim.Acta A 69 830

[29] Jingqun G, Bin L, Jun W, Xudong J, Renzheng J, Lijun L,Baoxin W and Yongnan X 2010 Spectrochim. Acta A 77 895

[30] Calzolai L, Franchini F, Gilliland D and Rossi F 2010 NanoLett. 10 3101

[31] Jang D J and Elsayed M A 1989 Proc. Natl. Acad. Sci. 86 5815[32] Jiang L, Yang B Q, Ma Y D, Liu Y C, Yang W S, Li T J and

Sun C C 2003 Chem. Phys. Lett. 380 29

[33] Machius M, Wiegand G and Huber R 1995 J. Mol. Biol.246 545

[34] Greenfield N J 2006 Natl. Protoc. 1 2527[35] Khosro K, Hossein N, Bijan R, Aliakbar M M and Mushin N

2001 Enzyme Microbial. Technol. 28 543[36] Boon L and Tee G K 2009 Biotechnol. Prog. 25 436[37] Kumari A and Kayastha A M 2011 J. Mol. Catal. B 69 8[38] Andrade M A, Chacon P, Merelo J J and Moran F 1993 Protein

Eng. 4 383

7

Biotechnology and Bioprocess Engineering 17: 000-000 (2012)

DOI 10.1007/s12257-011-0105-8

Immobilization of Porcine Pancreatic α-amylase on Magnetic Fe2O3

Nanoparticles: Applications to the Hydrolysis of Starch

Mohammad Jahir Khan, Qayyum Husain, and Ameer Azam

Received: 13 March 2011 / Revised: 30 December 2011 / Accepted: 20 January 2012

© The Korean Society for Biotechnology and Bioengineering and Springer 2012

Abstract Enzymes play a pivotal role in catalyzing diversereactions. However, their instability upon repetitive/pro-longed use, as well as their inhibition by high substratesand product concentration, remains an area of concern. Inthis study, porcine pancreatic α-amylase was immobilizedon magnetic Fe2O3 nanoparticles (Fe2O3-NPs) in order tohydrolyze starch. The magnetic nanoparticle bound enzymesretained 94% of their initial enzyme activity. X-ray diffr-action and atomic force microscopy analyses showed thatthe prepared matrix had advantageous microenvironmentand a large surface area for binding significant amounts ofprotein. Functional groups present in enzyme and supportwere monitored by Fourier transform infrared spectroscopy.Immobilized enzyme exhibited lowered pH optimum (pH6.0) to a greater degree than its soluble counterpart (pH7.0). Optimum temperature for the immobilized enzymeshifted towards higher temperatures. The immobilized enzymewas significantly more resistant to inactivation caused byvarious metal ions and chemical denaturants. Immobilizedα-amylase hydrolyzed 92% starch in a batch process, after8 h at 40oC; while the free enzyme could hydrolyze only73% starch under similar experimental conditions. Areusability experiment demonstrated that the immobilizedenzyme retained 83% of its original activity even after its

8th repeated use.

Keywords: α-Amylase, immobilization, nanoparticles,stabilization, starch hydrolysis

1. Introduction

The enzymes α-amylases (E.C. 3.2.1.1) catalyze thehydrolysis of α-1,4 glycosidic bonds present in starch,glycogen, and other related carbohydrates [1]. They arepresent in plants, animals, and microorganisms and haveextensive applications in medicine, textiles, fermentation,and the food industry [2]. The exploitation of solubleenzymes in industries is limited due to product inhibition,instability, non-reusability, and difficult recovery from areaction system [3]. In order to overcome these limitations,enzyme immobilization has been considered one of thebest alternatives to using enzymes on a large scale, inindustrial and environmental applications [4]. Immobili-zation also protects enzymes from denaturation and helpsto retain them in biochemical reactors in order to furthercatalyze the subsequent feed and to offer more economicaluse of biocatalysts in industry, waste water treatment, andthe development of bioprocess monitoring devices, such asbiosensors [5,6].

In the recent past, nanosized materials have been widelyemployed for enzyme immobilization. Due to the largesurface area of nanosized materials, they provide superiorloading capacity and low mass transfer resistance [7,8].The magnetic nanoparticles have now been used in con-junction with biological materials, such as proteins,enzymes, and nucleic acids, as they result in complete andeasy recovery of these materials from reaction systems,thereby reducing the operational costs of the processes [9-

Mohammad Jahir Khan, Qayyum HusainDepartment of Biochemistry, Faculty of Life Sciences, Aligarh MuslimUniversity, Aligarh 202-002, India

Qayyum Husain*

Faculty of Applied Medical Sciences, Jazan University, Jazan, Saudi ArabiaTel: 91-571-2700741; Fax: 91-571-2706002E-mail: [email protected]

Ameer AzamCentre of Excellence in Material Science (Nanomaterials), Zakir HusainCollege of Engineering and Technology, Aligarh Muslim University,Aligarh 202-002, India

RESEARCH PAPER

2 Biotechnology and Bioprocess Engineering 17: 000-000 (2012)

11].In the present work, an attempt has been made to immo-

bilize α-amylase on Fe2O3-NPs. The immobilized enzymewas characterized by X-ray diffraction (XRD), Fouriertransform-infrared (FT-IR) spectroscopy, and atomic forcemicroscopy (AFM). Thermal behavior of both soluble andimmobilized α-amylase was studied by thermo-gravimetric(TGA) and differential thermal analysis (DTA). Effects ofvarious metal ions and denaturing agents on the activity ofsoluble and immobilized α-amylase were monitored.Hydrolysis of starch by soluble and immobilized enzymeswas performed in batch processes. The reusability of theimmobilized enzyme was also examined.

2. Materials and Methods

2.1. Materials

Materials used included α-Amylase (Porcine pancreas),starch, maltose, glucose and 3,5 dinitro-salicylic acid (DNS);these were purchased from SRL Chemicals (Mumbai,India). Ferric nitrate, mono hydrated citric acid, and metalions were obtained from Sigma-Aldrich Co. (USA). Allother chemicals and reagents were of analytical grade andwere used without further purification.

2.2. Synthesis of Fe2O3-NP by sol-gel method

Ferric oxide nanoparticles (Fe2O3-NPs) were synthesizedaccording to the procedure described by Raming et al, withseveral modifications [12]. Ferric nitrate (200 mL, 0.1 M)was used as a precursor solution and was gelated with 800mL of mono hydrated citric acid (0.05 ~ 0.2 M); as ligandand triple distilled water were used as solvents. Ferricnitrate was added, dropwise, to the citric acid solution; itwas vigorously shaken. The solution was heated, at 70oC,with continuous stirring until the gel was formed and thewater was completely evaporated. The dried gel was thenannealed at 100oC for 16 h in order to solidify the gel,which was grounded for 30 min. The formed Fe2O3-NPwas used for further studies.

2.3. XRD analysis of Fe2O3-NP and Fe2O3-NP adsorbed

α-amylase

The crystal structure of Fe2O3-NP and Fe2O3-NP, whichadsorbed the α-amylase, was obtained with an X-raydiffractometer (Rigaku Miniflex X-ray diffractometer),using a monochromatized X-ray beam with nickel-filteredCu Kα radiation. A continuous scan mode was used tocollect 2 θ data from 5° to 80°. The particle size (D) of thesamples was determined by Scherer’s formula as:

D = 0.9 λ/B cos θ

where λ is the X-ray wavelength (1.54060 Å), B is the fullwidth at half-maximum of Fe2O3 (400) line, and θ is thediffraction angle.

2.4. Immobilization of α-amylase

Fe2O3-NP (40 mg) was added to α-amylase (2,083 U), andthis mixture was stirred overnight in 0.1 M sodium pho-sphate buffer, pH 7.0 at 4oC. The enzyme bound on nano-support was collected by centrifugation at 5,000 × g for 10min at 4oC. The unbound enzyme was removed by wash-ing it three times, with a 0.1 M sodium phosphate buffer,pH 7.0. The immobilized enzyme was stored in an assaybuffer at 4oC for further use.

2.5. AFM analysis

The tapping mode AFM experiment of free nanoparticlesand enzyme bound nanoparticles was performed usingcommercial etched silicon tips as an AFM probe, with acharacteristic resonance frequency of ca. 300 Hz (RTESP,Veeco). The samples were placed, dropwise, on a micawafer, air dried at room temperature for 12 h, and theimages were recorded with a Veeco Innova nanoscope IIAFM. Microscopic scans were carried out on several surfacepositions in order to check the surface uniformity.

2.6. FT-IR analysis of enzyme, Fe2O3-NP and Fe2O3-NP

adsorbed α-amylase

The FT-IR spectra of the enzyme, Fe2O3-NP and Fe2O3-NP, adsorbed α-amylase; these were recorded through thepotassium bromide pellet method on INTERSPEC 2020(USA) in the range of 400 ~ 4,000/cm. The calibration wasperformed by polystyrene film. Samples were injected witha Hamiet 100 µL syringe in an ATR box. The syringe waswashed with acetone; this was followed by distilled water.FT-IR analysis was performed in order to examine thefunctional groups present in the enzyme and support.

2.7. Thermogravimetric and differential thermal analysis

TGA was performed using a Mettler-3000 thermal analy-zer with a 2.0 mg sample, with heating rate of 10oC/min inN2 atmosphere. DTA analysis was also carried out in asimilar heating range, using TA-DSC, Q 200 (USA).

2.8. Effect of pH and temperature

Enzyme activity of soluble and immobilized α-amylase(51.0 U) was assayed in the buffers of different pHs (4.0 ~8.0). The buffers used were sodium acetate (pH 4.0, 5.0),sodium phosphate (pH 6.0, 7.0), and Tris-HCl (pH 8.0).The molarity of each buffer was 0.1 M. The activity at pH7.0 and pH 6.0 were taken as control (100%) for thecalculation of the remaining percentage of activity for thesoluble and immobilized enzymes, respectively.

Immobilization of Porcine Pancreatic α-amylase on Magnetic Fe2O3 Nanoparticles: Applications to the Hydrolysis of Starch 3

The activity of soluble and immobilized α-amylase (51.0U) was measured at various temperatures (20 ~ 80oC).Enzyme activity at 40 and 50oC was taken as control(100%) for the calculation of the remaining percentage ofactivity for the soluble and immobilized enzymes, respec-tively.

2.9. Effect of metal ions on activity of soluble and

immobilized α-amylase

Soluble and immobilized α-amylase (51.0 U) was in-dependently incubated, with a 5.0 mM solution of metalions (chlorides of Na+, K+, Mg2+, Cu2+, Co2+, Ba2+, Zn2+,Ca2+, and Fe3+); their activity was determined in a 0.1 Msodium phosphate buffer, pH 7.0 at 40oC. The activity ofenzymes in the absence of metal ions was taken as control(100%) for the calculation of the remaining percentage ofthe activity for the soluble and immobilized enzymes.

2.10. Effect of urea on activity of soluble and immobi-

lized α-amylase

Soluble and immobilized α-amylase (51.0 U) was incubatedwith a 4.0 M urea for different time intervals. The enzymeactivity without urea was taken as control (100%) for thecalculation of the remaining percentage activity for thesoluble and immobilized enzymes.

2.11. Reusability of immobilized α-amylase

Immobilized α-amylase (51.0 U) was taken in triplicates inorder to assay its activity. After each assay, the immobiliz-ed enzyme was taken out from assay tubes and stored in a0.1 M sodium phosphate buffer, pH 7.0 at 4°C. This proce-dure was repeated for eight consecutive days. The activitydetermined on first day was considered as the control(100%) for the calculation of the remaining percentageactivity after each use.

2.12. Hydrolysis of starch in batch process

Starch solution (1.0%, w/v) was incubated independently,with soluble and immobilized α-amylase (4,850 U) at 40oCwith constant stirring for 8 h. The aliquots (500 µL) weretaken out at indicated time intervals, hydrolysis of starchand the formation of maltose was assayed by the DNSmethod [13].

2.13. Activity assay for α-amylase

The activity of α-amylase was assayed by the DNS method[13], using a 1.0% soluble starch as a substrate. Theenzyme in a 0.1 M sodium phosphate buffer, pH 7.0 (250μL) and starch (250 μL), was incubated for 30 min at40°C. The reaction was stopped by adding 1.5 mL DNSreagent and heating the solution in boiling water for 5 min.DNS is a coloured reagent, and the reducing groupsreleased from starch by α-amylase action were measuredby the reduction of this reagent. After cooling, 1.5 mL ofdistilled water was added and, finally, absorbance wasrecorded spectrophotometrically, at 540 nm.

One unit (1.0 U) of α-amylase activity is defined as theamount of enzyme that liberates 1.0 µM of maltose permin, under standard assay conditions. A standard curve ofabsorbance against the amount of maltose was prepared tocalculate the amount of maltose released during assay.

2.14. Estimation of protein

Protein concentration was determined by a dye bindingmethod [14]. Bovine serum albumin was used as a standardprotein.

2.15. Statistical analysis

Each value represents the mean for three independentexperiments performed in duplicates, with average SDs of< 5%. Data were analyzed by one-way ANOVA, and p-values of < 0.05 were considered statistically significant.

3. Results and Discussion

3.1. Immobilization efficiency of α-amylase

Several methods have previously been employed for theimmobilization of α-amylase. Among these methods, phy-sical adsorption onto insoluble materials has been con-sidered one of the preferred choices, due to its simplicityand regeneration of support. Moreover, this method ofenzyme immobilization does not require pre-activationsteps for support [15].

Table 1 demonstrates the immobilization efficiency ofα-amylase. Fe2O3-NP adsorbed α-amylase preserved 94%of its initial enzyme activity which was higher compared toprevious reported immobilization studies [16,17].

3.2. XRD analysis

Table 1. α-amylase immobilized on Fe2O3-NP

Enzyme loaded(X) (U)

Enzyme activityin washes(Y) (U)

Activity bound to Fe2O3-NP (U)Activity yield (%)

(B/A × 100)Theoretical (X-Y=A)

Actual(B)

Effectiveness factor(B/A) = (η)

2,083 1,528 554 521 0.94 94


The diffraction patterns of Fe2O3-NP and Fe2O3-NP ad-sorbed α-amylase are represented in Fig. 1. The resultsshowed that nanoparticles have hexagonal crystal sym-metry with an average crystalline size of 26.90 nm (Fig.1A), which was decreased to 24.25 nm (Fig. 1B) afterenzyme adsorption. The intensity peaks of the NPs weremasked after immobilization as the enzyme covered signi-ficant parts of the NP surfaces [18]. The binding of mag-netic nanoparticles to bioactive substances involved anumber of interactions, including interactions betweenorganic ligands and those between amino acid side chainsof protein and metal centers. This might be accreditedbetter enzyme-substrate interactions on immobilization,with the availability of larger surface areas on nanoparticleand possible structural modulation or better exposure of theactive site [19]. Moreover, such coupling of biomolecularentities with nanoparticles provides enhanced thermal stabilityof the enzyme, allowing its use in various analytical, bio-technological, and industrial applications [20].

3.3. AFM and FT-IR analysis

Morphological information presents a physical picture ofhow α-amylase molecules are assembled on nanoparticlesurfaces (Fig. 2). The roughness shown in these images canalso be analyzed and related to the pattern of immobili-zation. We used the peak-to-valley distance in these imagesas an indicator of surface roughness. As shown (Fig. 2A),the nanoparticle surface is relatively smooth and has smallpeak-to-valley distance. Once an enzyme molecule is ad-sorbed (Fig. 2B), there was a significant increment in peak-to-valley distance. Thus, we can conclude that nanoparticlesurfaces were fully covered with enzymes after immobili-

zation, which corresponds to high enzyme loading [21,22].Fig. 3 shows FT-IR spectra of Fe2O3-NP, α-amylase and

Fe2O3-NP adsorbed α-amylase. FT-IR spectroscopy reveal-ed that the formation of hydrogen bonds between aminogroups and water may result in tremendous water solubi-lity, which makes them ideal for enzyme immobilization[23]. A sharp peak was observed at 538/cm (Figs. 3A and3C), which is characteristic of Fe-O vibrations. The amideI peak appeared between 1,600 and 1,700/cm due to C=Ostretching vibrations of the protein backbone. This peak issensitive to short range, imposed by distinct hydrogenbonding arrangements in the secondary structure of theproteins [24]. The peak at 1,108/cm (Fig. 3B) is most likelyreflecting an imidazole ring of the histidine side chain. Theinteractions of NPs with C-N groups of enzymes were alsoevident with the shifting of the amide A band from 3,341/cm toward ahigher frequency, 3,349/cm (Figs. 3B and 3C)[25].

3.4. TGA and DTA analysis

TGA and DTA results of free and Fe2O3-NP bound α-amylase are shown in Fig. 4. The TGA spectra of enzymeshowed a 5% loss in weight at 200°C, followed by a sharp

Fig. 1. XRD analysis of Fe2O3-NP and Fe2O3-NP adsorbed α-amylase. XRD analysis of Fe2O3-NP (A) and Fe2O3-NP adsorbedα-amylase (B) was obtained with an X-ray diffractometer (RigakuMiniflex X-ray diffractometer) using a monochromatized X-raybeam with nickel-filtered Cu Kα radiation, in the range of 5o ~80o.

Fig. 2. Atomic force microscopy of Fe2O3-NP and Fe2O3-NPadsorbed α-amylase. Tapping mode AFM experiments wereperformed using commercial etched silicon tips as AFM probeswith typical resonance frequencies of ca. 300 Hz. Atomic forcemicrographs for Fe2O3-NP (A) and Fe2O3-NP adsorbed α-amylase(B) have been shown.


peak at 400°C; this indicates a weight loss of over 65%(Fig. 4A). However, Fe2O3-NP adsorbed α-amylase ex-hibited very slight (5%) weight loss (Fig. 4C) under asimilar temperature range. The significant weight loss inthe free enzyme might be due to the removal of free andbound water and solvent.

The DTA curve showed that thermal decomposition ofenzymes begins at 180°C (Fig. 4B) while Fe2O3-NP ad-sorbed α-amylase retained a significantly higher stabilityof up to 300oC (Fig. 4D). This showed that the immobi-lized enzyme preparation can be employed at a highertemperature for various applications.

3.5. Effect of pH and temperature

The pH optimum for soluble enzymes was found at pH 7.0,which shifted to pH 6.0 upon immobilization (Fig. 5). Ashift in pH optimum upon immobilization depends onenzyme microenvironment as well as the structure andcharge of the matrix [26]. The change in pH optimum forimmobilized α-amylase has also been reported by severalearlier investigators [27,28].

The temperature optimum for α-amylase shifted from 40to 50oC after immobilization (Fig. 6). However, as thetemperature increased, activity of free enzymes decreasedmore rapidly, compared with the immobilized counterpart.The inability of a free enzyme to enhance its thermalstability is one of the major limitations for its application incontinuous reactors. An increase in temperature optimumof immobilized enzymes revealed that the enzyme mightbe more rigid to structural changes induced by heat [29,30].

Fig. 3. FT-IR spectra of Fe2O3-NP, α-amylase and Fe2O3-NP adsorbed α-amylase. FT-IR analysis was performed to monitor theinteractions between enzymes and nanoparticles. FT-IR spectra of Fe2O3-NP (A), α-amylase (B) and Fe2O3-NP adsorbed α-amylase (C)were monitored with INTERSPEC 2020 (USA) in the range of 400 ~ 4,000/cm.

Fig. 4. Thermogravimetric and differential thermal analysis of α-amylase and Fe2O3-NP adsorbed α-amylase. Fig. 4 shows athermogravimetric analysis of α-amylase (A) and Fe2O3-NP adsorb-ed α-amylase (C). The differential thermal analysis of α-amylase(B) and Fe2O3-NP adsorbed α-amylase has been shown in (D).


3.6. Effect of metal ions

Our studies showed that the activity of immobilized enzymeswas less affected in the presence of various metal ions,compared with free enzymes (Table 2). However, weobserved that Cu++ caused maximum inhibition in theactivity of both free and immobilized enzymes [31]. Therelative activity of the immobilized α-amylase was higherthan that of the soluble enzyme in the presence of NaCl,KCl, CaCl2, and CoCl2 [32].

3.7. Effect of urea

The immobilized α-amylase was more stable than thesoluble enzyme upon exposure to 4.0 M urea for a longerduration (Fig. 7). The result showed an 81% loss in enzymeactivity for soluble enzymes, while the immobilized enzymeretained more than 40% with identical exposure. Earlierstudies have indicated that proteins become unfolded dueto direct interaction of urea molecules with the peptidebackbone, via hydrogen bonding or hydrophobic interactions[33]. Molecular crowding/confinement theory has predictedthat Fe2O3-NPs provide a beneficial confined space thatresists conformational changes to the immobilized enzyme,even at higher concentration of urea [34].

Fig. 5. pH activity profiles for soluble and immobilized α-amylase. The enzyme activity of soluble and immobilized α-amylase (51.0 U) was measured in the buffers of various pHs. Thebuffers used were: sodium acetate (pH 4.0, 5.0), sodiumphosphate (pH 6.0, 7.0) and Tris-HCl (pH 8.0). The molarity ofeach buffer was 0.1 M. The activity at pH 7.0 and pH 6.0 weretaken as controls (100%) for the calculation of remaining percentactivity for soluble and immobilized α-amylase, respectively. Thesymbols show (- -) soluble and (- -) immobilized α-amylase.□ ■

Table 2. Effect of metal ions on soluble and immobilized α-amylase

Metal ions(5.0 mM)

Remaining activity (%)a

Immobilized enzyme Soluble enzyme

None 100 100

NaCl 106.9 ± 2.90 118.1 ± 1.71

KCl 103.6 ± 1.11 110.5 ± 2.51

MgCl2 96.3 ± 0.97 97.6 ± 2.85

CuCl2 21.7 ± 2.00 35.7 ± 2.37

CoCl2 81.4 ± 1.94 114.7 ± 1.43

BaCl2 112.2 ± 2.54 91.4 ± 1.65

ZnCl2 53.0 ± 1.27 77.6 ± 1.25

CaCl2 97.5 ± 1.11 105.7 ± 0.95

FeCl3 63.1 ± 2.46 92.3 ± 1.69

aThe percent activity of an enzyme compared with the activity withoutthe presence of metal ions.

Fig. 6. Temperature activity profiles for soluble and immobilizedα-amylse. The activity of soluble and immobilized α-amylase(51.0 U) was measured at various temperatures (20 ~ 80oC) for 30min. The enzyme activity at 40 and 50oC was taken as control(100%) for the calculation of remaining percent activity for thesoluble and immobilized enzymes, respectively. For symbols,please refer to figure legend 5.

Fig. 7. Effect of 4.0 M urea on soluble and immobilized α-amylase. Soluble and immobilized α-amylase (51.0 U) wasincubated with 4.0 M urea, in a 0.1 M sodium phosphate buffer,pH 7.0. Enzyme activity was determined at different time intervalsunder conditions mentioned in the text. To calculatethe remainingpercent activity, untreated samples were considered as controls(100%). For symbols, please refer to figure legend 5.


3.8. Hydrolysis of starch in batch processes by soluble

and immobilized α-amylase

Table 3 describes the hydrolysis of starch by the solubleand immobilized α-amylase for various time intervals at40oC. The soluble and immobilized enzymes hydrolyzed61 and 68% starch after 4 h of incubation, respectively.Additionally, the maximum hydrolysis of starch, 74% bythe free enzyme, was attained after 8 h, while the immobi-lized enzyme hydrolyzed a higher concentration of starch(92%) under similar incubation time. This was due to thefact that the soluble enzyme was more accessible to thesubstrate for first few hours but, after a prolonged period of

time, the rate of hydrolysis decreased more quickly. Thismight be due to the enzyme unfolding or the inhibition ofenzyme activity by its own product [35,36]. Satish et al.

reported similar results, where starch was hydrolyzed by α-amylase immobilized on super porous cellulose beads [37].

3.9. Reusability

Enzymes are quite expensive products, so reusability andoperational stability are important criteria for enzymeapplication at the industrial level. Immobilized α-amylaseshowed 83% residual activity even after the 8th consecutiveuse (Fig. 8). The significant retention in immobilized enzymeactivity on repeated uses might be due to the multipointnon-covalent attachment of the enzyme to nanosupport.

4. Conclusion

In this study, α-amylase was immobilized onto Fe2O3-NPsby a simple adsorption mechanism with excellent catalyticefficiency. Immobilized α-amylase was found to be stableagainst various types of physical and chemical denaturants.The increased temperature optimum of immobilizedenzyme results in several benefits; higher temperaturedecreased the viscosity of both substrate and product,increased the reaction rate with less operational time, andminimized the risk of microbial contamination. Reusabilityexperiments further supported that the adsorbed enzymedid not detach from the nanosupport with repeated use and,therefore, such preparations could be considered for thecontinuous hydrolysis of starch for longer durations, evenat higher temperatures. Thus, such immobilized enzymescould prove useful for the efficient conversion of starch incontinuous reactors at the industrial level.

Acknowledgement

Aligarh Muslim University, Aligarh is gratefully acknow-ledged for providing UGC, scholarship to one of us (M. J.Khan).

Nomenclature

AFM : Atomic force microscopyDNS : 3,5 Dinitro-salicylic acidDTA : Differential thermal analysisFT-IR : Fourier transform-infrared spectroscopyNPs : NanoparticlesTGA : Thermo-gravimetric analysisXRD : X-ray diffraction

Table 3. Hydrolysis of starch by soluble and immobilized α-amylase in batch processes at 40oC

Time (min) Remaining activity (%)

Soluble Immobilized

0 ND ND

30 14 ± 0.69 9 ± 1.39

60 20 ± 2.11 15 ± 1.42

90 25 ± 1.34 20 ± 2.48

120 36 ± 2.08 42 ± 0.99

180 47 ± 1.22 59 ± 2.22

240 61 ± 1.34 68 ± 1.90

300 67 ± 0.98 74 ± 1.10

360 73 ± 1.75 83 ± 0.79

420 73 ± 1.11 92 ± 1.15

480 73 ± 2.43 92 ± 1.42

ND: Not determined.

Fig. 8. Reusability of immobilized α-amylase. The reusability ofimmobilized α-amylase was monitored for 8 successive days. Thepreparation was taken in triplicates and assayed for the remainingpercentage activity. The activity determined on the first day wastaken as the control (100%) for the calculation of the remainingpercentage activity after each use.


References

1. Bordbar, A. K., K. Omidiyan, and R. Hosseinzadeh (2005) Studyon interaction of α-amylase from Bacillus subtilis with cetyl tri-methyl ammonium bromide. Colloid. Surf. B: Bioint. 40: 67-71.

2. Abd El-Ghaffar, M. A. and M. S. Hashem (2009) Immobilizationof α-amylase onto chitosan and its amino acid condensationadducts. J. Appl. Polym. Sci. 112: 805-814.

3. Akoh, C. C., S. W. Chang, G. C. Lee, and J. F. Shaw (2008) Bio-catalysis for the production of industrial products and functionalfoods from rice and other agricultural produce. J. Agric. FoodChem. 56: 10445-104451.

4. Husain, Q. (2010) β Galactosidases and their potential applica-tions. Crit. Rev. Biotechnol. 30: 41-62.

5. Husain, Q. (2006) Potential applications of the oxidoreductiveenzymes in the decolorization and detoxification of textile andother synthetic dyes from polluted water: A review. Crit. Rev.Biotechnol. 26: 201-221.

6. Mateo, C., O. Abian, R. Fernandez-Lafuente, and J. M. Guisan(2000) Increase in conformational stability of enzymes immobi-lized on epoxy-activated supports by favoring additional multi-point covalent attachment. Enz. Microb. Technol. 26: 509-515.

7. Chen, B., M. E. Miller, and R. A. Gross (2007) Effects of porouspolystyrene resin parameters on Candida antarctica lipase Badsorption, distribution, and polyester synthesis activity. Lang-muir 23: 6467-6474.

8. Kim, M. I., J. Kim, J. Lee, and H. Jia (2007) Crosslinked enzymeaggregates in hierarchically-ordered mesoporous silica: A simpleand effective method for enzyme stabilization. Biotechnol.Bioeng. 96: 210-218.

9. Safarik, I. and M. Safarikova (2009) Magnetic nano and micro-particles in biotechnology. Chem. Pap. 63: 497-505.

10. Safarikova, M., L. Ptackova, I. Kibrikova, and I. Safar k (2005)Biosorption of water-soluble dyes on magnetically modified Sac-charomyces cerevisiae subsp. uvarum cells. Chemosphere 59:831-835.

11. Namdeo, M. and S. K. Bajpai (2009) Immobilization of α-amy-lase onto cellulose-coated magnetite (CCM) nanoparticles andpreliminary starch degradation study. J. Mol. Catal. B: Enzym.59: 134-139.

12. Raming, T. P., A. J. Winnubst, C. M. van Kats, and A. P. Philipse(2002) The synthesis and magnetic properties of nanosizedhematite (α-Fe2O3) particles. J. Colloid Int. Sci. 249: 346-350.

13. Bernfield, P. (1955) Amylases α and β. pp. 149-158. In: S. P.Colowick and N. O. Kaplan (eds.). Methods in enzymol. Aca-demic press, NY.

14. Bradford, M. M. (1976) Rapid and sensitive method for quanti-tation of microgram quantities of protein utilizing principle ofprotein-dye binding. Anal. Biochem. 72: 248-254.

15. Iyer, P. V. and L. Ananthanarayan (2008) Enzyme stability andstabilization-aqueous and non-aqueous environment. Proc. Bio-chem. 43: 1019-132.

16. Kennedy, J. F. and M. Paterson (1993) Application of cellulosicfast flow column filters to protein immobilization and recovery.Polym. Int. 32: 71-81.

17. Hasirci, N., S. Aksoy, and H. Tumturk (2006) Activation of poly(dimmer acid-co-alkyl polyamine) particles for covalent immo-bilization of α-amylase. Reac. Func. Polym. 66: 1546-1551.

18. Mallikarjuna, N. N., S. K. Manohar, P. V. Kulkarni, A. Venkat-araman, and T. M. Aminabhavi (2005) Novel high dielectric con-stant nanocomposites of polyaniline dispersed with γ-Fe2O3

nanoparticles. J. Appl. Polym. Sci. 97: 1868-1874.19. Weng, L., L. Zhang, D. Ruan, L. Shi, and J. Xu (2004) Thermal

gelation of cellulose in a NaOH/thiourea aqueous solution. Lang-muir 20: 2086-2093.

20. Konwarh, R., N. Karak, S. K. Rai, and A. K. Mukherjee (2009)Polymer-assisted iron oxide magnetic nanoparticle immobilizedkeratinase. Nanotechnol. 20: 225107-225117.

21. Scaramuzzo, F. A., R. Salvati, B. Paci, A. Generosi, V. Rossi-Albertini, A. Latini, and M. Barteri (2009) Nanoscale in situ mor-phological study of proteins immobilized on gold thin films. J.Phys. Chem. B. 113: 15895-15899.

22. Saal, K., V. Sammelselg, A. Lohmus, E. Kuusk, G. Raidaru, T.Rinken, and A. Rinken (2002) Characterization of glucose oxi-dase immobilization onto mica carrier by atomic force micros-copy and kinetic studies. Biomol. Eng. 19: 195-199.

23. Lei, C. H., Y. Shin, J. Liu, and E. J. Ackerman (2002) Entrappingenzyme in a functionalized nanoporous support. J. Am. Chem.Soc. 124: 11242-11243.

24. Jackson, M. and H. H. Mantsch (1995) The use and misuse ofFTIR spectroscopy in the determination of protein structure. Crit.Rev. Biochem. Mol. Biol. 30: 95-120.

25. Krimm, S. and J. Bandekar (1986) Vibrational spectroscopy andconformation of peptides, polypeptides and proteins. Adv. ProteinChem. 38: 181-364.

26. Roig, M. G, A. Slade, and J. F. Kennedy (1993) α-Amylaseimmobilized on plastic supports: Stabilities, pH and temperatureprofiles and kinetic parameters. Biomat. Artif. Cells Immob. Bio-technol. 21: 487-525.

27. Bayramoglu, G., M. Yilmaz, and M. Y. Arica (2004) Immobili-zation of thermostable α-amylase onto reactive membranes:Kinetics characterization and application to continuous starchhydrolysis. Food Chem. 84: 591-599.

28. Arica, M. Y., V. Hasirci, and N. G. Alaeddinoglu (1995) Covalentimmobilization of α-amylase onto pHEMA microspheres: Prep-aration and application to fixed bed reactor. Biomaterials 16:761-768.

29. Magri, M. L., M. V. Miranda, and O. Cascone (2005) Immobili-zation of soybean seed coat peroxidase on polyaniline: Synthesisoptimization and catalytic properties. Biocatal. Biotrans. 23:339-346.

30. Lee, P. M., K. H. Lee, and S. Y. Siaw (1993) Covalent immobi-lization of aminoacrylase to alginate for L-phenylalanine produc-tion. J. Chem. Technol. Biotechnol. 58: 65-70.

31. Ramesh, M. V. and B. K. Lonsane (1990) Effect of metal saltsand protein modifying agents on activity of thermostable α-amy-lase produced by Bacillus lichiniformis M27 under solid state fer-mentation. Chem. Microb. Technol. Lebensm. 12: 129-136.

32. Lo, H., L. Lin, H. Chen, W. Hsu, and C. Chang (2001) Enzymaticproperties of a SDS-resistant Bacillus sp. TS-23 α-amylase pro-duced by recombinant Escherichia coli. Proc. Biochem. 36: 743-750.

33. Makhatadze, G. I. and P. L. Privalov (1992) Protein interactionswith urea and guanidinium chloride. A calorimetric study. J. Mol.Biol. 226: 491-505.

34. Zhou, H. X. (2004) Protein folding and binding in confinedspaces and in crowded solutions. J. Mol. Recognit. 17: 368-375.

35. Tanriseven, A. and S. Dogan (2002) A novel method for theimmobilization of β-galactosidase. Proc. Biochem. 38: 27-30.

36. Yagar, H., F. Ertan, and B. Balkan (2008) Comparison of someproperties of free and immobilized α-amylase by Aspergillussclerotiorum in calcium alginate gel beads. Prep. Biochem. Bio-technol. 38: 13-23.

37. Shewale, S. D. and A. B. Pandit (2007) Hydrolysis of solublestarch using Bacillus licheniformis α-amylase immobilized onsuperporous CELBEADS. Carbohyd. Res. 342: 997-1008.

i

Application of Immobilized Ipomoea batata β Amylase in the Saccharification of Starch Mohammad Jahir KHAN Fahim Halim KHAN Qayyum HUSAIN*Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh (202002), INDIA

*Corresponding Author e-mail: [email protected]

AbstractPresent study demonstrates the immobilization of acetone fractionated Ipomoea batata (sweet potato) β amylase on an inorganic support,

Celite-545 by simple adsorption mechanism. The adsorbed enzyme exhibited an activity yield of 244 U g-1 of the matrix with effectiveness factor ‘η’ 0.83. Interaction between Celite-545 and enzyme was confirmed by fourier transform infrared spectroscopy and atomic force microscopy. The binding efficiency of enzyme to the support was analyzed by eluting it with 1.0 M NaCl. Both soluble and immobilized β amylase exhibited same pH optima while temperature optimum of immobilized enzyme was shifted from 50oC to 60oC. The immobilized β amylase preparation was superior to the free enzyme in hydrolyzing starch in a batch process: it hydrolyzed starch to 88% and 96% at 40oC and 50oC, respectively whereas soluble enzyme hydrolyzed only 83% and 80% of starch under similar experimental conditions. The immobilized β amylase retained 84% of its original activity after 30 days storage at 4oC, while the soluble enzyme showed only 41% of the initial activity under identical conditions. Immobilized β amylase retained 79% activity even after its 7th repeated use.

Keywords: β amylase; Celite-545; immobilization; sweet potato; batch processAbbreviations: AFM, atomic force microscopy; DNS, 3,5 dinitro-salicylic acid; FT-IR, fourier transform infrared spectroscopy

INTRODUCTION

β Amylase (E.C. 3.2.1.2) is an exoamylase which is widely distributed in higher plants and microorganisms. Among plant sources sweet potato is thought to be a promising source of β amylase with fair thermostability [1]. It catalyzes the hydrolysis of α-1, 4 glycosidic linkages at non reducing end of starch and related carbohydrates [2]. The biotechnological application of β amylase includes the production of maltose and high maltose syrups. Maltose is widely applied in food and pharmaceutical industries, since its properties are represented by mild sweetness, good thermal stability and low viscosity in solution [3,4].

An effective application of free enzymes is hindered due to several drawbacks like thermal instability, rapid loss of catalytic activity during operation and storage period and sensitivity to numerous denaturing agents [5,6]. Such drawbacks can be circumvented by using enzymes in their immobilized form [7-9].

Several methods including entrapment, adsorption, encapsulation in membranes, chemical cross linking by using bifunctional or multifunctional reagents and bioaffinity based procedures have been employed previously for enzyme immobilization [10,11]. Among these methods, adsorption of enzymes on particulate carriers is one of the simplest and cost effective immobilization techniques [12].

Celite-545 has recently been used as an immobilization matrix owing its inexpensive nature and other desirable physical

Journal of Applied Biological Sciences 5 (2): 33-39, 2011ISSN: 1307-1130, E-ISSN: 2146-0108, www.nobel.gen.tr

properties like large surface area with high enzyme loading and enormous porosity which increase enzyme accessibility to the substrate. Moreover, it shows higher thermal and chemical stabilities with greater resistance to microbial degradation [13,14].

In this study, I. batata β amylase has been immobilized on an inexpensive support, Celite-545. The immobilized enzyme preparations were characterized by fourier transform infrared spectroscopy (FT-IR) and atomic force microscopy (AFM) in order to monitor the functional groups and surface topography, respectively. The stability of soluble and immobilized β amylase has been investigated against several physical and chemical denaturants. Hydrolysis of starch in batch processes at varying temperatures by soluble and immobilized enzyme was also evaluated.

MATERIALS AND METHODS

MaterialsCelite-545 (20-45 µ mesh) was obtained from Serva

Labs (Heidelberg, Germany). Starch, maltose, glucose and DNS were purchased from SRL Chemicals (Mumbai, India). Acetone was obtained from Merck (Darmstadt, Germany). I. batata was purchased from local market. Other chemicals and reagents employed were of analytical grade and used without any further purification.

34 M. J. Khan et al / JABS, 5 (2): 33-39, 2011

Extraction and partial purification of β amylase from sweet potato

Mature, healthy Ipomoea batata roots (200 g) were washed thoroughly with distilled H2O, cut into small pieces and homogenized in 100 ml of 0.1 M sodium phosphate buffer, pH 6.0. The suspension was filtered through 4 layers of cheesecloth. Filtrate thus obtained was centrifuged at 10 000 x g for 30 min at 4oC. The crude extract was initially fractioned by 50% (v/v) chilled acetone saturation. This solution was continuously stirred overnight at 4oC for complete precipitation of proteins. After centrifugation at 10 000 x g for 30 min, precipitated pellets were collected and resuspended in two-pellet volume of cold buffer. The solution was dialyzed against 0.1 M phosphate buffer of pH 6.0 for overnight. Undissolved particles were removed by centrifugation and the clear solution was stored in assay buffer at 4oC for further use.

Adsorption of β amylase on Celite-545Celite-545 (1.0 g) was suspended in 50 ml of 0.1 M

phosphate buffer and stirred for 1 h at room temperature. The fine particles present in suspension were removed by decantation and similar procedure was repeated thrice [15]. The binding of β amylase on support was carried out by incubating 1232 U of enzyme g-1 of Celite-545 and this mixture was stirred overnight in 0.1 M sodium phosphate buffer, pH 6.0 at 4oC. The enzyme bound on Celite-545 was collected by centrifugation at 3 000 x g for 15 min at 4oC. Unbound enzyme was removed by washing thrice with buffer and immobilized enzyme was stored in assay buffer at 4oC for further use.

FT-IR spectra of Celite-545 and Celite-545 adsorbed β amylase

FT-IR spectra of Celite-545 and Celite-545 adsorbed β amylase were recorded by the potassium bromide pellet method on INTERSPEC 2020 (USA) in the range of 400-4000 cm-1. The calibration was done by polystyrene film. The samples were injected by Hamiet 100 µl syringe in ATR box. The syringe was first washed with acetone followed by distilled water. FT-IR analysis was done to examine the functional groups present in enzyme and support.

AFM analysisTapping mode AFM experiments of Celit-545 and Celite-545

adsorbed β amylase were performed using commercial etched silicon tips as AFM probes with typical resonance frequency of ca. 300 Hz (RTESP, Veeco). The samples were placed drop wise on a mica wafer, air dried at room temperature for 12 h and the images were recorded with a Veeco Innova nanoscope II AFM. AFM scans were carried out on several surface positions to check the surface uniformity.

Effect of NaCl on immobilized enzymeThe Celite-545 adsorbed β amylase (1.0 U) was incubated

with 1.0 M NaCl in 0.1 M sodium phosphate buffer, pH 6.0 at 50 oC for varying times. Activity of untreated enzyme was considered as control (100%) for the calculation of remaining percent activity.

Effect of pHSoluble and immobilized β amylase (1.0 U) was assayed in

the buffers of different pH (pH 2.0-8.0). The buffers used were glycine-HCl (pH 2.0), sodium acetate (pH 3.0, 4.0), sodium phosphate (pH 5.0-7.0) and tris-HCl (pH 8.0). The molarity of each buffer was 0.1 M. Maximum activity obtained at pH 6.0 was taken as control (100%) for the calculation of remaining percent activity.

Effect of temperatureEffect of temperature on soluble and immobilized β

amylase (1.0 U) was studied by measuring activity of enzyme preparations at various temperatures (20-80oC) in 0.1 M sodium phosphate buffer, pH 6.0.

In another set of experiment, soluble and immobilized β amylase (1.0 U) was independently incubated at 60°C in 0.1 M sodium phosphate buffer, pH 6.0, for varying times. Aliquots of each preparation were taken at indicated time intervals, chilled quickly in ice for 5 min and activity was measured. The activity obtained without incubation at 60°C was taken as control (100%) for the calculation of remaining percent activity.

Storage stability Soluble and the immobilized β amylase were stored at 4oC

in 0.1 M sodium phosphate buffer, pH 6.0 for over 30 days. The aliquots from each preparation (1.0 U) were taken in triplicates at the gap of 5 days and were then analyzed for the remaining enzyme activity. The activity determined on the first day was taken as control (100%) for the calculation of remaining percent activity.

Reusability of immobilized β amylaseImmobilized enzyme was taken in triplicates for assaying

β amylase activity. After each assay the immobilized enzyme preparation was taken out, washed, and stored overnight in 0.1 M sodium phosphate buffer, pH 6.0, at 4°C. The activity was assayed for seven successive days. The activity determined for the first day was considered as control (100%) for the calculation of remaining percent activity after each use.

Starch hydrolysis in batch processStarch solution (1% w/v) was independently incubated

with soluble and immobilized β amylase (500 U) at 50oC and 60oC respectively under stirring condition for 6 h. Aliquots were taken out at different time intervals and assayed for the formation of maltose by DNS method [16].

Measurement of β amylase activityActivity of β amylase was assayed by DNS method with

slight modifications [16]. 250 µl of enzyme in buffer was added to 250 µl substrate (1% w/v) and the resulting mixture was incubated for 30 min at 50oC. Reaction was stopped by adding 1.5 ml of DNS solution and then heated in a boiling water bath for 5 min. After cooling, reaction mixture was diluted with distilled water. Absorbance was measured spectrophotometrically at 540 nm with maltose as standard.

35M. J. Khan et al / JABS, 5 (2): 33-39, 2011

One unit (1.0 U) of β amylase activity is defined as the amount of enzyme that liberating 1 mg of maltose min-1 under the standard assays conditions. A standard curve of absorbance against amount of maltose was constructed to calculate the amount of maltose released during assay.

Estimation of proteinProtein concentration was estimated according to the

procedure described by Lowry et al [17]. BSA was used as a standard protein.

Statistical analysisEach value represents the mean for three independent

experiments performed in duplicates, with average SDs, <5%. Data were analyzed by one-way ANOVA. P-values <0.05 were considered statistically significant.

RESULTS AND DISCUSSION

Immobilization efficiency of β amylase on Celite-545The present study involves direct immobilization of partially

purified β amylase from I. batata on an inexpensive support, Celite-545. Thus, the cost of enzyme purification is minimized. Celite-545 is an inorganic mechanically stable, non-toxic and non-biodegradable diatomaceous earth which has been used widely to immobilize various enzymes and proteins [15,18]. The binding of β amylase on support was significantly affected by change in pH. Enzyme was maximally adsorbed at pH 6.0 and retained 244 U β amylase activity g-1 of Celite-545 with 83% preserved activities (Table 1). In the literature, for immobilized β amylase, various values for binding capacities and preserved activities are given. For example, when immobilization of sweet potato β amylase was achieved on chitosan beads and spheron based support, preserved activities were reported as 59% and 76%, respectively[19,20]. Furthermore 49% immobilized efficiency was observed on immobilizing barley β amylase on polyacrylamide polymer [21].

FT-IR analysisThe FT-IR spectra of Celite-545 and Celite-545 adsorbed β

amylase are used to investigate the interaction between enzyme and support (Fig. 1). The premier intensity peak at 1079.63 cm-1

is due to asymmetric Si-O-Si stretching vibration of Celite-545. The band at 791.57 cm-1 is attributed to the symmetric stretching of the ring structure of (SiO)4 tetrahedra [22]. The vibration of H2O caused by the hydrogen bonds of protein with silanol groups is presented at 1639.48 cm-1 [23]. Furthermore, peak at 3362.70 cm-1 due to C-H stretching indicated strong interactions of enzyme with Celite-545 [24]. Intensity peak of Si-O-Si stretching vibration at 1079.63 cm-1 was decreased to 1074.20 cm-1 when enzyme was adsorbed to support surface.

AFM analysisVisualization of surface topography of support and enzyme

adsorbed on it with AFM revealed a significant amount of enzyme molecules immobilized on the support (Fig. 2). The functional groups existing on Celite-545 surface were also verified by FT-IR spectroscopy. We used the peak-to-valley distance in these images as an indicator of the surface roughness. The support surface, before β amylase immobilization, was smooth (Fig. 2a), compared with the enzyme immobilized surfaces. It is evident that as the immobilization progressed the roughness of support surface increases, which could be seen from the increase of peak-to-valley value (Fig. 2b). The roughness of the sample surface is an important feature and should play an important role in affecting the enzyme activity. This observation is consistent with those in previous reports [25].

Effect of 1.0 M NaCl on immobilized β amylase The activity of immobilized enzyme was evaluated after

incubating it with 1.0 M NaCl for 4 h at 50oC (Fig. 3). The adsorbed enzyme exhibited retention of signifcant enzyme activity even in presence of 1.0 M NaCl. Result showed that binding of β amylase with Celite-545 was quite strong and such type of immobilized enzyme preparations could be easily exploited for industrial applications. Ashraf and Husain, showed similar results with radish peroxidase when immobilized on DEAE cellulose[26].

Enzyme Loaded (X) (U)

Enzyme activityin washes (Y) (U)

Activity bound/g of celite 545 (U) Activity yield % (B/A×100)

1232 939

Theoretical (X-Y=A)

Real (B)

Effectiveness factor(B/A) = (η)

83% 292 244 0.83

Table 1. β Amylase Immobilized on Celite-545

Each value represents the mean for three independent experiments performed in duplicates, with average standard deviations, < 5%.

Figure. 1. FT-IR spectra of Celite-545 (a) and Celite-545 adsorbed β amylase (b)

791.

57

1079

.63

1074

.2016

39.4

8

2359

.81

3362

.70

70

72

74

76

78

80

82

84

86

88

90

92

94

96

98

100

%T

1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1)

(a)

(b)

36 M. J. Khan et al / JABS, 5 (2): 33-39, 2011

Effect of pH A shift in enzyme activity upon immobilization towards

acidic or basic directions is natural since the microenvironment of the free and immobilized enzyme is quite different. Charge and structure of support material, along with nature of the activators impart a significant effect on enzyme activity. Therefore, comparing the activity of soluble and immobilized enzyme as a function of pH forms an important part of the study [27].

Fig. 4 demonstrates the pH-activity profiles for soluble and immobilized β amylase. Both soluble and immobilized enzyme preparations showed the same pH-optima, pH 6.0. However, immobilized β amylase retained significantly higher enzyme activity at alkaline sides as compared to soluble under similar experimental conditions. Several earlier investigators have previously reported the broadening in pH optima for the immobilized amylases [18,28].

Effect of temperatureThe inability to enhance the thermal stability of a native

enzyme is one of the most important limitations for their application in continuous reactors. Studies showed an increase in temperature-optimum from 50oC to 60oC for immobilized enzyme (Fig. 5). At 70oC the activity retained by immobilized enzyme was significantly higher (83%) as compared to soluble enzyme (28%). Thermal inactivation studies showed 88% of the initial activity retained by immobilized enzyme after 2 h exposure at 60oC whereas the free enzyme exhibited 62%

activity under identical thermal exposure (Fig 5). Further incubation of soluble enzyme at 60oC for 4 h resulted in a loss of 64% activity whereas immobilized enzyme retained significantly higher activity, 66%.

Noda et al in their studies observed higher temperature optima for immobilized β amylase in comparison to its soluble countepart18. Similarly an increase in temperature-optima was noticed when soybean β amylase was immobilized on chitosan beads. The shift in temperature optima of immobilized enzyme might be due to conformational changes at higher temperatures that might expose active sites more appropriate for substrate interaction thereby increasing its apparent enzymatic activity [29,30].

Reusability of immobilized β amylaseEnzymes are quite expensive products. Immobilization as a

technique ensures the recycling and reusability of the enzyme. The most important advantage of immobilization is its repeated uses. Reusability pattern of immobilized β amylase showed about 79% of initial activity retention even after its 7th repeated use (Fig. 7). The trivial activity loss upon reuse could be due to frequent encountering of substrate into the same active site which might distort it and this distortion could dwindle the catalytic efficiency either partially or fully[31].

Figure 2. 3D AFM images of Celite-545 (a) and Celite-545 β amylase (b)

Figure 3. Effect of ions (NaCl) on immobilized β amylase

Figure 4. pH-activity profiles of soluble and immobilized β amylase

(a)

(b)

90

92

94

96

98

100

102

104

0 30 60 90 120 150 180 210 240 270

Time (min)

Rel

ativ

e ac

tivity

(%)

10

30

50

70

90

110

1 2 3 4 5 6 7 8 9 10pH

Rel

ativ

e ac

tivity

(%)

37M. J. Khan et al / JABS, 5 (2): 33-39, 2011

Starch hydrolysis in batch processesStarch is a very important raw material for the production

of sweeteners, adhesives, thickening and binding agents. Table 3 illustrates the hydrolysis of starch in batch process by soluble and immobilized β amylase at 40oC and 50oC, respectively. It was noticed that the hydrolysis of starch by soluble enzyme was 82% after 5 h at 40oC whereas immobilized enzyme exhibited 88% of the hydrolytic activity under identical conditions.

Hydrolysis of starch with free and immobilized β amylase at 50oC after 3 h incubation was 74% and 85% respectively. It was observed that only 80% starch was hydrolyzed by the free enzyme after 4 h at 50oC. However, the hydrolysis of starch by soluble enzyme beyond this limit did not exhibit any significant increase whereas the maximum starch hydrolysis achieved by immobilized enzyme reached to 96% in 6 h (Table 3). The results showed that the rate of hydrolysis was more in case of soluble enzyme for the first few hours as compared to immobilized enzyme. It was due to the fact that soluble enzyme was more accessible to starch for first few hours but after prolonged incubation, the rate of hydrolysis decreased. It might be due to enzyme unfolding or inhibition of enzyme activity by its own product [32,33]. Satish et al have reported similar results where starch was hydrolyzed by immobilized α-amylase on super porous CELBEADS [34].

Figure 5. Temperature activity profiles of soluble and immobilized β amylase

Figure 6. Thermal denaturation of soluble and immobilized β amylase

Figure 7. Reusability of immobilized β amylase

0

20

40

60

80

100

120

10 20 30 40 50 60 70 80 90Temperature

Rel

ativ

e ac

tivity

(%)

0

20

40

60

80

100

120

0 30 60 90 120 150 180 210 240 270Time (min)

Rel

ativ

e ac

tivity

(%0

0

20

40

60

80

100

120

1 2 3 4 5 6 7Number of uses

Rel

ativ

e ac

tivity

(%)

Series3

Storage stabilityTable 2 depicts the storage stability of soluble and

immobilized β amylase. Immobilized enzyme retained 84% of its initial activity after 30 days of storage while its soluble counterpart exhibited only 43% of its original enzyme activity. The greater stability offered by this inexpensive immobilized β amylase preparation for longer duration and at higher temperatures might bring about its use in the continuous production of novel products of industrial importance at large scale.

Table 2. Storage stability of soluble and immobilized β amylase at 4oC

Soluble and the immobilized β amylase were stored at 4 oC in 0.1 M sodium acetate buffer, pH 6.0 for over 30 days. The aliquots from each preparation were taken in triplicates at the gap of 5 d and were then analyzed for the remaining enzyme activity. Each value represents the mean for three independent experiments performed in duplicates, with average standard deviation, < 5%.

Number of days Remaining activity (%)

SβA IβA Control 100 100

05 91 ± 1.41 97 ± 1.68

10 77 ± 1.25 94 ± 1.57

15 60 ± 1.13 93 ± 0.97

20 52 ± 2.31 88 ± 1.22

25 47 ± 1.88 85 ± 2.10

30 43 ± 1.09 84 ± 1.32

SβA; Soluble β amylase IβA; Immobilized β amylase

38 M. J. Khan et al / JABS, 5 (2): 33-39, 2011

CONCLUSION

Amylases are among the most important enzymes used for industrial purposes, and now in the light of biotechnology they are considered useful for biopharmaceutical applications. Here, an attempt has been made to obtain a simple, inexpensive and stable immobilized β amylase. The proteins were directly immobilized by adsorption from the crude homogenate, thus avoiding the high cost of enzyme purification. The immobilized β amylase exhibited better thermostability than the free enzyme which resulted in several benefits including low viscosity of substrates and products, minimized bacterial contaminations, increased reaction rates and decrease of operation time. Furthermore, immobilized enzyme significantly hydrolyzed starch in batch processes at high temperatures. Thus the reactors containing such types of inexpensive immobilized enzyme preparations could be exploited for the continuous hydrolysis of starch at large scale.

AcknowledgementThe authors are thankful to University Grants Commission,

New Delhi, India, for providing special grant DRS, SAP to the department for developing infrastructural facility

REFERENCES

[1] Kossam J, Lloyd J. 2000. Understanding and influencing starch biochemistry. Crit Rev Biochem Mol Biol. 35: 141-196.

[2] Bryjak J. 2003. Glucoamylase, α amylase and β amylase immobilization on acrylic carriers. Biochem Eng J. 16: 347-355.

[3] Ramachandran S, Patel AK, Nampoothiri KM, Chandran S, Szacaks G, Soccol CR, Pandey A. 2004. α Amylase

from a fungal culture grown on oil cakes and its properties, Brazil Arch Biol Technol. 47: 309-317

[4] Ertan F, Yagar H, Balkan B. 2006. Some properties of free and immobilized α-amylase from Penicillium griseofulvum by solid state fermentation. Prep Biochem Biotechnol. 36: 81-91.

[5] Sivaramakrishnan S, Gangadharan D, Madhavan NK, Soccol CR, Pandey A. 2006. α Amylases from microbial sources; An overview on recent developments. Food Technol Biotechnol. 44: 173-184.

[6] Giorno L, Molinari R, Drioli E, Bianchi D, Cesti P. 2004. Performance of a biphasic organic/aqueous hollow fibre reactor using immobilized lipase. J Chem Technol Biotechnol. 64: 345-532.

[7] Iyer V, Ananthanarayan L. 2008. Enzyme stability and stabilization-aqueous and non-aqueous environment. Process Biochem. 43: 1019-1032.

[8] Husain Q. 2010. β galactosidase and their potential applications: A review. Crit Rev Biotechnol. 30: 41-62.

[9] Carpio C, Batista VF, Ruales J. 2009. Improved glucoamylase immobilization onto calcined chicken bone particles. Food Bioproc Technol. 9: 214-219.

[10] Reshmi R, Sanjay G, Sugunan S. 2006. Enhanced activity and stability of α amylase immobilized on alumina, Catal Commun. 7: 460-465.

[11] Haider T, Husain Q. 2007. Calcium alginate entrapped preparations of Aspergillus oryzae β galactosidase: Its stability and applications in the hydrolysis of lactose. Int J Biol Macromol. 41: 72-80.

[12] Lee D H, Kang S G, Suh S G, Byun J K. 2003. Purification and characterization of β galactosidase from peach (Prunus persica). Mol Cells, 15: 68-74.

[13] Satar R, Husain Q. 2009. Adsorption of peroxidase on Celite-545 directly from ammonium sulphate fractionated white radish (Raphanus sativus) proteins. Biotechol J. 4: 408-416.

[14] Matto M, Husain Q. 2009. Decolorization of textile effluent by bitter gourd peroxidase immobilized on concanavalin A layered calcium alginate-starch beads. J Hazard Mat. 164: 1540-1546.

[15] Khan A A, Akhtar S, Husain Q. 2006. Direct immobilization of polyphenol oxidases on Celite 545 from ammonium sulphate fractionated proteins of potato (Solanum tuberosum). J Mol Catal B: Enzym. 40: 58-63.

[16] Bernfield P. 1955. Amylases α/β. Methods enzymol, Academic press, New York, 149-158.

[17] Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. 1957. Protein measurements with the folin phenol reagent. J Biol Chem. 193: 265-275.

[18] Xu L, Feng YQ. 2004. Applications of ordered mesoporous materials in separation science. Chin J Anal Chem. 32: 374-380.

[19] Noda T, Furuta S, Suda I. 2001. Sweet potato β amylase immobilized on chitosan beads and its application in the semi-continous production of maltose. Carb Polym. 44: 189-195.

Table 3. Hydrolysis of starch by soluble and immobilized β amylase in batch process at different temperatures

Starch hydrolysis was performed as described in text. Each value represents the mean for three independent experiments performed in duplicates, with average standard deviations, < 5%.

Time (min) RA% (at 40o

C) RA% (at 50o

C)

SβA IβA SβA IβA

0 ND ND ND ND

30 59 ± 1.21 46 ± 1.71 52 ± 2.20 49 ± 1.20

60 64 ± 1.53 53 ± 2.44 56 ± 2.63 54 ± 1.74

90 67 ± 1.33 59 ± 1.22 63 ± 1.87 60 ± 1.09

120 67 ± 1.88 64 ± 0.79 64 ± 1.24 72 ± 2.15

150 72 ± 2.34 70 ± 1.57 69 ± 1.88 78 ± 1.99

180 81 ± 1.09 82 ± 1.63 74 ± 1.51 85 ± 1.53

240 82 ± 0.97 85 ± 2.09 80 ± 0.99 92 ± 1.41

300 82 ± 1.86 88 ± 1.11 80 ± 0.83 96 ± 2.00

360 82 ± 1.14 88 ± 2.34 80 ± 1.29 96 ± 1.57

RA; Remaining activityND; Not determined

39M. J. Khan et al / JABS, 5 (2): 33-39, 2011

[20] Gray CJ, Lee CM, Barker SA. 1982. Immobilization of enzyme on spheron: 2. β Amylase, the development of a column reactor separator. Enzyme Microb Technol. 4: 147-153.

[21] Atia KS, Ismail SA, Dessouki AM. 2003. Immobilization of β amylase using polyacrylamide polymer derivatives. J Chem Technol Biotech. 78: 891-898.

[22] Evans CR, Spurlin TA, Frey BL. 2002. In situ FT-IR measurements of competitive vapor adsorption into porous thin films containing silica nanoparticles. Anal Chem. 74: 1157.

[23] Prakasham RS, Devi GS, Laxmi KR, Rao CS. 2007. Novel synthesis of ferric impregnated silica nanoparticles and their evaluation as a matrix for enzyme immobilization. J Phys Chem. 111: 3842-3847

[24] Yu JH, Du YM, Zheng H. 1999. Blend films of chitosan-gelation. J Wuhan Univ, (Nal. Sci. Ed.) 45: pp 440-444.

[25] Djambaski1 P, Aleksieva P, Spasova D, Chernev G, Nacheva L. 2010. Immobilization in nanomatrices of humicola lutea mycelium for alpha-galactosidase biosynthesis in laboratory air-lift bioreactor. Biotechnol Biotechnol. 24: 1897-1903.

[26] Ashraf H, Husain Q. 2010. Stabilization of DEAE cellulose adsorbed and glutaraldehyde crosslinked white radish ( Raphanus sativus) peroxidase. j Sci Indus Res. 69: 613-620.

[27] Aksoy S, Tumturk H, Hasirci N. 1998. Stability of α amylase immobilized on poly (methyl methacrylate-acrylic acid) microspheres. J Biotechnol. 60: 37-46.

[28] Kahraman MV, Bayramoglu G, Kayaman-Apohan N, Gungor A. 2007. α-Amylase immobilization on functionalized glass beads by covalent attachment. Food Chem. 104: 1385-1392.

[29] Yoshida M, Oishhi K, Ogata M & Nakakuki T. 1989. Continous production of maltose using a dual immobilized enzyme system. Agric Biol Chem. 53: 3139-3142.

[30] Anderson WA, Reilly PM, Moo YM, Legge RL. 2009. Application of a bayesian regression method to the estimation of diffusivity in hydrophilic gels. Can J Chem Eng. 70: 499-504

[31] Konosoula Z, Liakopoulou-Kyriakides M. 2006. Starch hydrolysis by the action of an entrapped in alginate capsules α-amylase from Bacillus subtilis. Proc Biochem. 41: 343-349.

[32] Yagar H, Ertan F, Balkan B. 2008. Comparison of some properties of free and immobilized α amylase by Aspergillus sclerotiorum in calcium alginate gel beads. Prep Biochem. 38: 13-23.

[33] Tanriseven A, Dogan S. 2002. A novel method of immobilization of β galactosidase, Process Biochem. 36: 27-30.

[34] Shewale SD, Pandit AB. 2007. Hydrolysis of soluble starch using Bacillus licheniformis α amylase immobilized on superporous CELBEADS. Carb Res. 342: 997-1008.

List of publications and presentations -...

Documents

Transcript of List of publications and presentations -...