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Magnetic immobilization and characterization of α-amylase as nanobiocatalyst for hydrolysis of sweet potato starch
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Research article
Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch G. Baskar ∗ , N. Afrin Banu, G. Helan Leuca, V. Gayathri, N. Jeyashree Department of Biotechnology, St. Joseph’s College of Engineering, Chennai 600 119, India
a r t i c l e
i n f o
Article history: Received 2 December 2014 Received in revised form 6 February 2015 Accepted 12 February 2015 Available online xxx Keywords: Immobilisation Amylase Characterization Enzyme biocatalysis Sweet potato starch Enzyme kinetics
a b s t r a c t Magnetic immobilization of enzymes became emerging method for efficient recovery of biocatalyst under magnetic field. Sweet potato (Ipomoea batatas) root starch is abundantly produced in tropical countries. Thus the present work was focused on immobilizing ␣-amylase on to magnetic nanoparticles and use as biocatalyst for hydrolysis of sweet potato starch into glucose. The hydrolysis of sweet potato starch using immobilized ␣-amylase was investigated and the maximum glucose yield of 42.89 mg/g was obtained using 3% (w/v) of sweet potato starch concentration with an initial pH 4 at 40 ◦ C in 80 min reaction time. The optimal concentration of immobilized ␣-amylase for maximum glucose yield was found as 1% (w/v). The kinetics of hydrolysis reaction was studied using Michaelis–Menten equation. The sweet potato starch was found to have more affinity towards magnetically immobilized ␣-amylase with substrate affinity constant (Km) of 0.16 mg/ml and the maximum reaction rate (Vmax ) of 3.63 × 10−3 mol/ml s. The magnetically immobilized nanocomposite of ␣-amylase can be easily recovered and reused for maximum utilization. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Fossil fuels are a finite energy resource and we can portend the depletion of these resources in a few decades. Biofuels are energy sources derived from biological materials that distinguishes them from other non-fossil fuel energy sources such as wind, hydro and wave energy [1,2]. We have been on the man-hunt for cheaper and renewable energy resources since the dawn of this century. Starch is one the best and most high yielding feedstock for ethanol production, hydrolysis is required to produce ethanol from starch by fermentation [3–5]. Starch was traditionally hydrolyzed using acids as catalyst. The major disadvantages of acid hydrolysis of starch include the possible inhibitory effect of by-products, neutralization of hydrolyzates before fermentation, and the expensive construction material for equipment. Such drawbacks have estimated enzyme based hydrolysis superior to conventional acid based hydrolysis [6,7]. The specificity of enzymes as catalyst toward the substrate, their inherent mild reaction conditions, and the absence of secondary reactions has made the amylase as preferred catalyst for starch hydrolysis in to starch hydrolysate [8–10]. In today’s scenario there is an immense attraction toward nanoparticles and its application in catalysis, due to their unique
large surface-to-volume ratio and quantum size effects [11]. The preparation of metal nanoparticles can be classified into physical and chemical methods. Nanoparticles prepared by chemical methods have usually a narrow size distribution when compared to physical methods [12,13]. Developing a novel method of immobilizing enzymes as biocatalyst is important for easy recovery and reuse in industrial biotechnology. Thus the present work was focused on optimization process parameters and kinetics of hydrolysis of sweet potato starch using nanobiocomposite of ␣-amylase immobilized on to magnetic nanoparticles. The immobilized ␣amylase was recovered under magnetic field and reused.
2. Materials and methods 2.1. Chemicals and biochemicals Fungal ␣-amylase of Aspergillus oryzae was purchased from HiMedia, Mumbai India. Dinitro salicylic acid, Gluteraldehyde, FeSO4 and FeCl3 were purchased from SRL Pvt., Ltd., Mumbai, India.
2.2. Sweet potato starch preparation ∗ Corresponding author. Tel.: +91 9443678571; fax: +91 4424500861. E-mail address: [email protected] (G. Baskar).
Sweet potato is the largest vegetable crop grown in tropical countries. The root of sweet potato was sliced into small pieces
http://dx.doi.org/10.1016/j.bej.2015.02.020 1369-703X/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: G. Baskar, et al., Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch, Biochem. Eng. J. (2015), http://dx.doi.org/10.1016/j.bej.2015.02.020
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Fig. 1. (a) MNP suspension (b) MNP under magentic field (c) amylase solution (d) amylase immoblized on pretreated MNP (e) amylase immbilized MNP under magentic field.
and dried. The dried slices were ground in to powder and dried at 80 ◦ C overnight. 2.3. Magnetic immobilization of ˛-amylase enzyme Magnetic nanoparticle was prepared by co-precipitation of FeSO4 and FeCl3 solution. 50 ml of 0.2 M FeCl3 and 50 ml of 0.1 M FeSO4 solutions were prepared and mixed together. The solution was kept under constant mixing with drop wise addition of 50 ml of 10% NaOH to the mixture until black precipitate was obtained. The resultant precipitate was magnetically decanted and dried in oven at 80 ◦ C. The dried magnetic nanoparticles and ␣-amylase were mixed in the ratio of 1:1 in phosphate buffer (pH 4) and kept in a shaker for 30 min. Gluteraldehyde (1%, v/v) was added in to the mixture of enzyme-magnetic nanoparticle and continued in the shaker for 2 h. Then the magnetically immobilized ␣-amylase was separated by magnetic decantation [14]. 2.4. Characterization of immobilized ˛-amylase using FTIR, SEM with EDS The magnetic effect of both MNP and MNP with amylase was confirmed using a strong magnet. The structure, size, shape and elemental composition of the synthesized magnetically immobilized ␣-amylase were analyzed using field emission scanning electron microscope (FESEM) from CARL ZEISS, Germany and energy dispersive spectroscope (EDS) from Oxford Instruments, United Kingdom. The phase structure of the nanocatalyst was studied by X-ray diffraction analysis (XRD) from Rikagu, Japan. 2.5. Batch studies on hydrolysis of sweet potato starch using immobilized ˛-amylase The effect of sweet potato starch concentration on hydrolysis by magnetically immobilized ␣-amylase was studied under fixed other conditions. The starch concentration was varied from 2.5% to 6% (w/v) with an interval of 0.5%. The effect of initial pH (3–8) of the starch solution on hydrolysis was studied under fixed other conditions. The effect of reaction temperature (25, 30, 35, 40 and 45 ◦ C) and magnetically immobilized ␣-amylase concentration (0.2, 0.4, 0.6, 0.8 and 1.0%(w/v)) on hydrolysis of sweet potato starch were studied under fixed other conditions. The reusability of
magnetically immobilized ␣-amylase for hydrolysis of sweet potato starch was studied under constant conditions. 2.6. Estimation of glucose The glucose concentration in sweet potato starch hydrolysate was estimated using Dinitro Salicylic Acid (DNSA) method. The samples collected from reaction mixture were mixed with DNS reagent. Then the reaction mixture was mixed well and capped in order to avoid evaporation and boiled in water bath for 10 min. Then reaction mixture was cooled to room temperature and absorbance was observed at 540 nm by using spectrophotometer [15]. 3. Results and discussion 3.1. Magentic separation of immobilized ˛-amylase on magnetic nanoparticles The magnetic effect of MNP and MNP with amylase was confirmed using a strong magnet as shown in Fig. 1. The MNP suspension in Fig. 1(a) was strongly attracted by a magnet as shown in Fig. 1(b). The MNP was completely accumulated nearer to the magnet. The ␣-amylase immobilized MNP in Fig. 1(d) was separated under the influence of magnetic field as shown in Fig. 1(e). Thus the magnetically immobilized ␣-amylase can be effectively separated and reused. 3.2. Activity and specific activity of immobilized enzyme The activity and specific activity of MNP immobilized ␣-amylase were found to be 8.65 U/ml and 9.95 U/mg, respectively. The activity and specific activity of free enzyme were found to be 13.22 U/ml and 11.26 U/mg, respectively. The immobilized amylase has retained 88.37% of its original activity as compared to free enzyme. The amylase loading on MNP was found to be 173.91 mg/g. 3.3. Characterization of immobilized ˛-amylase using SEM, EDS, FTIR and XRD The surface characteristics of magnetically immobilized ␣amylase were studied using FE-SEM as shown in Fig. 2. The shape of magnetic nanocomposite of ␣-amylase was confirmed as spherical. The size of the nanocomposite was found to vary from 51.58 nm to
Please cite this article in press as: G. Baskar, et al., Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch, Biochem. Eng. J. (2015), http://dx.doi.org/10.1016/j.bej.2015.02.020
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Fig. 2. SEM analysis of magnetic nanocomposite of ␣-amylase.
21.10 nm and the average particle size was 37.18 nm. The surface of the nanocomposite was found porous in nature which might have enhanced the catalytic activity of nanocomposite of ␣-amylase. Hence the immobilized of ␣-amylase can be recovered from reaction mixture using by the action of magnetic field. The elemental composition of the nanocompoiste was studied using EDS analysis as shown in Fig. 3. The presence of iron, oxygen and carbon was confirmed with the help of EDS analysis. Thus the magnetic effect of nanocomposite of ␣-amylase was confirmed. The functional group present on the surface of nanocomposite of ␣-amylase was analyzed using FTIR as shown in Fig. 4(a). The peaks were observed at 3773.4, 3432.8, 2927.8, 2345.9, 1688.1, 1410.1 and 1101.5. The peak at 3773.4 and 3432.8 represents the presence of N H group with strong primary amine and weak to
moderate secondary amine which confirms the presence of protein (␣-amylase) in magnetic nanocomposite. The peak at 2927.8 represents moderate to strong alkyl bond and peak at 1688.1 represents the C O bond which confirms the presence of aromatic carboxylic acids in nanocomposite. The peak at 1410.1 also represents the presence of weak to strong aromatic C C bond. The presence of alkyl bond and aromatic carboxylic acids might be due to the use of glutaraldehyde as conjugating agent. The results of XRD analysis of magnetic nanocomposite of ␣-amylase is shown in Fig. 4(b). The high peaks were observed at 2 values of 35.56, 57.20 and 62.76. The peak at 35.56 represents diffraction plane (3 1 1), 57.20 corresponds to diffraction plane (5 1 1) and 62.76 corresponds to diffraction plane (4 4 0). Thus the magnetic nanocomposite of ␣amylase was found to be crystalline and cubical in structure. The
Fig. 3. EDS analysis of magnetic nanocomposite of ␣-amylase.
Please cite this article in press as: G. Baskar, et al., Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch, Biochem. Eng. J. (2015), http://dx.doi.org/10.1016/j.bej.2015.02.020
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Fig. 4. (a) FTIR spectrum (b) XRD analysis of magnetic nanocomposite of ␣-amylase.
fungal ␣-amylase from A. oryzae was reported to have three dimensional crystalline structures [16]. Thus the ␣-amylase immobilized on to magnetic nanparticles by simple adsorption, retained its original structure. 3.4. Hydrolysis of sweet potato starch using immobilized ˛-amylase The effect of sweet potato starch concentration on hydrolysis using magnetically immobilized ␣-amylase nanocomposite was observed as shown in Fig. 5(a). The glucose yield was found to increase with increase in starch concentration from 2.5 to 3% (w/v) and reaction time. The decrease in glucose yield was observed for further increase in starch concentration from up to 6%. The amylase activity and percentage conversion of sweet potato starch were found to increase with increases in starch concentration. However the glucose yield was found to decreases with increases in starch concentration. This might be due to the relative decrease in percentage conversion of sweet potato starch into glucose. The maximum glucose yield of 42.56 mg/g was obtained for 3% starch concentration in 80 min of reaction time. The effect of initial pH on hydrolysis of sweet potato starch using magnetically immobilized nanocomposite of ␣-amylase was observed as shown in Fig. 5(b). The initial pH of sweet potato starch solution was varied from 3 to 8 to study its effect on glucose yield. The glucose yield was found to increase with increase in initial pH from 3 to 4 and reaction time. The decrease in glucose yield was observed when the initial pH was further increased from 3 to 8. The maximum glucose yield of 41.34 mg/g was obtained for initial pH 4 in 100 min of reaction time. The effect of reaction temperature on hydrolysis of sweet potato starch was studied by varying the temperature from 25 to 45 ◦ C with an interval of 5 ◦ C as shown in Fig. 5(c). The increase in temperature from 35 to 40 ◦ C was found to increase the glucose yield with increase in reaction time. Then the glucose yield was decreased due
to further increase in temperature to 45 ◦ C. The maximum glucose yield of 41.34 mg/g was obtained in 80 min of reaction time at 40 ◦ C. The effect of varied concentration of nanocomposite of ␣-amylase was studied as shown in Fig. 5(d). The increase in glucose yield was observed for increase in nanocomposite concentration from 0.2 to 1%(w/v). The maximum glucose yield of 41.28 mg/g was obtained in 120 min reaction time using 1% nanocomposite concentration. The immobilized ␣-amylase was recovered using magnetic field and reused for hydrolysis of sweet potato starch (Fig. 6). The immobilized ␣-amylase was found to be active during reuse and 75% its initial hydrolytic activity was obtained even after fifth cycle. 3.5. Kinetics of sweet potato starch hydrolysis by immobilized ˛-amylase The substrate dependency of enzymatic hydrolysis of sweet potato starch was studied in terms of kinetics. The MichaelisMenten kinetics of the enzymatic reactions is given by Eq. (1).
v=
vmax [s] Km + [s]
(1)
where V is the reaction rate, Km is the Michaelis–Menten constant, Vmax is the maximum reaction rate and [S] is the substrate concentration. The Lineweaver–Burk plot is an efficient graphical method of determination of Michaelis–Menten parameters. The Lineweaver–Burk plot provides a more precise way to determine Vmax and Km . The Lineweaver–Burk plot was described by Lineweaver and Burk in 1934 [17,18]. The Michaelis–Menten parameters such as substrate affinity constant (Km ) and substrate saturation constant (Vmax ) obtained from Lineweaver–Burk plot with a correlation co-efficient of 0.9976 (R2 ) were 0.16 mg/ml and 3.63 × 10−3 mol/ml s, respectively. Thus, the magnetically immobilized ␣-amylase has shown high affinity toward sweet potato starch.
Please cite this article in press as: G. Baskar, et al., Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch, Biochem. Eng. J. (2015), http://dx.doi.org/10.1016/j.bej.2015.02.020
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Fig. 5. Optimization of hydrolysis conditions (a) effect of sweet potato starch concentration (♦2.5%; 3.0%; 3.5%; ×4.0%; *4.5%; 䊉5.0%; |5.5%; --6%) (b) Effect of initial pH (♦3; 4; 5; ×6; *7; 䊉8) (c) Effect of reaction temperature (♦25 ◦ C; 30 ◦ C; 35 ◦ C; ×40 ◦ C; *45 ◦ C) (d) effect of magnetically immobilized amylase concentration (♦0.2%; 0.4%; 0.6%; ×0.8%; *1.0%).
in 80 min of reaction time at 40 ◦ C. Thus magnetically immobilized nanocomposite of ␣-amylase can be effectively recovered and reused for maximum utilization thereby decrease in cost production of starch hydrolysate. References
Fig. 6. Relative percentage activity and glucose yield of reused magnetically immobilized ␣-amylase.
4. Conclusions The fungal ␣-amylase enzyme was efficiently immobilized on magnetic nanoparticle and used for hydrolysis of sweet potato starch into glucose. The maximum glucose yield of concentration of 42.89 mg/g was obtained using 3% sweet potato starch with an initial pH of 4 and 1% magnetic nanobiocomposite of ␣-amylase
[1] H.S. Alan, Biofuels: Production, Application and Development, 1st ed., CABI, 2010. [2] G. Vijai Kumar, G.T. Maria, Biofuel Technologies: Recent Developments, Springer, 2013. [3] S.R. Purohit, B.K. Mishra, Simultaneous saccharification and fermentation of overnight soaked sweet potato for ethyl alcohol fermentation, Adv. J. Food Sci. Technol. 4 (2012) 56–59. [4] S.M. Tiquia-Arashiro, M. Mormile, Sustainable technologies: bioenergy and biofuel from biowaste and biomass, Environ. Technol. 34 (13–16) (2013) 1637–1638. [5] C. Lareo, M.D. Ferrari, M.G. Guigou, L.F. Fajardo, V. Larnaudie, M.B. Ramirez, J.M. Garreiro, Evaluation of sweet potato for fuel bioethanol production: hydrolysis and fermentation, Springerplus 2 (2013) 493. [6] D.Y. Corredor, J.M. Salazar, K.L. Hohn, S. Bean, B. Bean, D.D. Wang, Evaluation and characterization of forage Sorghum as feedstock for fermentable sugar production, Appl. Biochem. Biotechnol. 158 (2009) 164–179. [7] K. Theerarattananoon, X. Wu, A. Staggenborg Scott, R. Propheter, R. Madl, D. Wang, Evaluation and characterization of sorghum biomass as feedstock for sugar production, J. Trans. ASABE 53 (2010) 509–525. [8] T. Baba, T.K. Kainuma, Partial hydrolysis of sweet-potato starch with -amylase, Agric. Biol. Chem. 51 (1987) 1365–1371. [9] A.M. Omemu, I. Akpan, M.O. Bankole, O.D. Teniola, Hydrolysis of raw tuber starches by amylase of Aspergillus niger AM07 isolated from the soil, Afr. J. Biotechnol. 4 (2005) 19–25. [10] G. Baskar, C. Muthukumaran, S. Renganathan, Optimization of enzymatic hydrolysis of manihot esculenta root starch by immobilized ␣-amylase using response surface methodology, Int. J. Chem. Biomol. Eng. 1 (2008) 155–159.
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[11] T. Naoki, Nanoscale Materials, Metal nanoparticles for catalysis, 2003, Chapter. 3. [12] B.R. Cuenya, Synthesis and catalytic properties of metal nanoparticles: size, shape, support, composition, and oxidation state effects, Thin Solid Films 518 (2010) 3127–3150. [13] M. Ahmadipour, M. Hatami, K. Venkateswara Rao, Preparation and characterization of nano-sized (Mgx Fe1−x O/SiO2x=0.1 core–shell nanoparticles by chemical precipitation method, Adv. Nanoparticles 1 (2012) 25166. [14] Y.F. Shen, J. Tang, Z.H. Nie, Y.D. Wang, Y. Ren, L. Zuo, Preparation and application of magnetic Fe3 O4 nanoparticles for wastewater purification, Sep. Purif. Technol. 68 (2009) 312–319.
[15] P. Trinder, Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor, Ann. Clin. Biochem. 6 (1969) 24–27. [16] E.A. Mac Gregor, Alpha-amylase structure and activity, J. Protein Chem. 7 (1988) 399–415. [17] H. Lineweaver, D. Burk, The determination of enzyme dissociation constants, J. Am. Chem. Soc. 56 (1934) 658–666. [18] G. Baskar, S. Arulluca Sundarsingh, S. Bharath Kumar, C. Muthukumaran, S. Renganathan, Optimization and kinetics of enzymatic hydrolysis of cassava starch by cross-linked fungal ␣-amylase, Asian, J. Microbiol. Biotechnol. Environ. Sci. 11 (2009) 323–326.
Please cite this article in press as: G. Baskar, et al., Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch, Biochem. Eng. J. (2015), http://dx.doi.org/10.1016/j.bej.2015.02.020
ARTICLE IN PRESS
BEJ-6132; No. of Pages 6
Biochemical Engineering Journal xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Research article
Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch G. Baskar ∗ , N. Afrin Banu, G. Helan Leuca, V. Gayathri, N. Jeyashree Department of Biotechnology, St. Joseph’s College of Engineering, Chennai 600 119, India
a r t i c l e
i n f o
Article history: Received 2 December 2014 Received in revised form 6 February 2015 Accepted 12 February 2015 Available online xxx Keywords: Immobilisation Amylase Characterization Enzyme biocatalysis Sweet potato starch Enzyme kinetics
a b s t r a c t Magnetic immobilization of enzymes became emerging method for efficient recovery of biocatalyst under magnetic field. Sweet potato (Ipomoea batatas) root starch is abundantly produced in tropical countries. Thus the present work was focused on immobilizing ␣-amylase on to magnetic nanoparticles and use as biocatalyst for hydrolysis of sweet potato starch into glucose. The hydrolysis of sweet potato starch using immobilized ␣-amylase was investigated and the maximum glucose yield of 42.89 mg/g was obtained using 3% (w/v) of sweet potato starch concentration with an initial pH 4 at 40 ◦ C in 80 min reaction time. The optimal concentration of immobilized ␣-amylase for maximum glucose yield was found as 1% (w/v). The kinetics of hydrolysis reaction was studied using Michaelis–Menten equation. The sweet potato starch was found to have more affinity towards magnetically immobilized ␣-amylase with substrate affinity constant (Km) of 0.16 mg/ml and the maximum reaction rate (Vmax ) of 3.63 × 10−3 mol/ml s. The magnetically immobilized nanocomposite of ␣-amylase can be easily recovered and reused for maximum utilization. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Fossil fuels are a finite energy resource and we can portend the depletion of these resources in a few decades. Biofuels are energy sources derived from biological materials that distinguishes them from other non-fossil fuel energy sources such as wind, hydro and wave energy [1,2]. We have been on the man-hunt for cheaper and renewable energy resources since the dawn of this century. Starch is one the best and most high yielding feedstock for ethanol production, hydrolysis is required to produce ethanol from starch by fermentation [3–5]. Starch was traditionally hydrolyzed using acids as catalyst. The major disadvantages of acid hydrolysis of starch include the possible inhibitory effect of by-products, neutralization of hydrolyzates before fermentation, and the expensive construction material for equipment. Such drawbacks have estimated enzyme based hydrolysis superior to conventional acid based hydrolysis [6,7]. The specificity of enzymes as catalyst toward the substrate, their inherent mild reaction conditions, and the absence of secondary reactions has made the amylase as preferred catalyst for starch hydrolysis in to starch hydrolysate [8–10]. In today’s scenario there is an immense attraction toward nanoparticles and its application in catalysis, due to their unique
large surface-to-volume ratio and quantum size effects [11]. The preparation of metal nanoparticles can be classified into physical and chemical methods. Nanoparticles prepared by chemical methods have usually a narrow size distribution when compared to physical methods [12,13]. Developing a novel method of immobilizing enzymes as biocatalyst is important for easy recovery and reuse in industrial biotechnology. Thus the present work was focused on optimization process parameters and kinetics of hydrolysis of sweet potato starch using nanobiocomposite of ␣-amylase immobilized on to magnetic nanoparticles. The immobilized ␣amylase was recovered under magnetic field and reused.
2. Materials and methods 2.1. Chemicals and biochemicals Fungal ␣-amylase of Aspergillus oryzae was purchased from HiMedia, Mumbai India. Dinitro salicylic acid, Gluteraldehyde, FeSO4 and FeCl3 were purchased from SRL Pvt., Ltd., Mumbai, India.
2.2. Sweet potato starch preparation ∗ Corresponding author. Tel.: +91 9443678571; fax: +91 4424500861. E-mail address: [email protected] (G. Baskar).
Sweet potato is the largest vegetable crop grown in tropical countries. The root of sweet potato was sliced into small pieces
http://dx.doi.org/10.1016/j.bej.2015.02.020 1369-703X/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: G. Baskar, et al., Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch, Biochem. Eng. J. (2015), http://dx.doi.org/10.1016/j.bej.2015.02.020
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Fig. 1. (a) MNP suspension (b) MNP under magentic field (c) amylase solution (d) amylase immoblized on pretreated MNP (e) amylase immbilized MNP under magentic field.
and dried. The dried slices were ground in to powder and dried at 80 ◦ C overnight. 2.3. Magnetic immobilization of ˛-amylase enzyme Magnetic nanoparticle was prepared by co-precipitation of FeSO4 and FeCl3 solution. 50 ml of 0.2 M FeCl3 and 50 ml of 0.1 M FeSO4 solutions were prepared and mixed together. The solution was kept under constant mixing with drop wise addition of 50 ml of 10% NaOH to the mixture until black precipitate was obtained. The resultant precipitate was magnetically decanted and dried in oven at 80 ◦ C. The dried magnetic nanoparticles and ␣-amylase were mixed in the ratio of 1:1 in phosphate buffer (pH 4) and kept in a shaker for 30 min. Gluteraldehyde (1%, v/v) was added in to the mixture of enzyme-magnetic nanoparticle and continued in the shaker for 2 h. Then the magnetically immobilized ␣-amylase was separated by magnetic decantation [14]. 2.4. Characterization of immobilized ˛-amylase using FTIR, SEM with EDS The magnetic effect of both MNP and MNP with amylase was confirmed using a strong magnet. The structure, size, shape and elemental composition of the synthesized magnetically immobilized ␣-amylase were analyzed using field emission scanning electron microscope (FESEM) from CARL ZEISS, Germany and energy dispersive spectroscope (EDS) from Oxford Instruments, United Kingdom. The phase structure of the nanocatalyst was studied by X-ray diffraction analysis (XRD) from Rikagu, Japan. 2.5. Batch studies on hydrolysis of sweet potato starch using immobilized ˛-amylase The effect of sweet potato starch concentration on hydrolysis by magnetically immobilized ␣-amylase was studied under fixed other conditions. The starch concentration was varied from 2.5% to 6% (w/v) with an interval of 0.5%. The effect of initial pH (3–8) of the starch solution on hydrolysis was studied under fixed other conditions. The effect of reaction temperature (25, 30, 35, 40 and 45 ◦ C) and magnetically immobilized ␣-amylase concentration (0.2, 0.4, 0.6, 0.8 and 1.0%(w/v)) on hydrolysis of sweet potato starch were studied under fixed other conditions. The reusability of
magnetically immobilized ␣-amylase for hydrolysis of sweet potato starch was studied under constant conditions. 2.6. Estimation of glucose The glucose concentration in sweet potato starch hydrolysate was estimated using Dinitro Salicylic Acid (DNSA) method. The samples collected from reaction mixture were mixed with DNS reagent. Then the reaction mixture was mixed well and capped in order to avoid evaporation and boiled in water bath for 10 min. Then reaction mixture was cooled to room temperature and absorbance was observed at 540 nm by using spectrophotometer [15]. 3. Results and discussion 3.1. Magentic separation of immobilized ˛-amylase on magnetic nanoparticles The magnetic effect of MNP and MNP with amylase was confirmed using a strong magnet as shown in Fig. 1. The MNP suspension in Fig. 1(a) was strongly attracted by a magnet as shown in Fig. 1(b). The MNP was completely accumulated nearer to the magnet. The ␣-amylase immobilized MNP in Fig. 1(d) was separated under the influence of magnetic field as shown in Fig. 1(e). Thus the magnetically immobilized ␣-amylase can be effectively separated and reused. 3.2. Activity and specific activity of immobilized enzyme The activity and specific activity of MNP immobilized ␣-amylase were found to be 8.65 U/ml and 9.95 U/mg, respectively. The activity and specific activity of free enzyme were found to be 13.22 U/ml and 11.26 U/mg, respectively. The immobilized amylase has retained 88.37% of its original activity as compared to free enzyme. The amylase loading on MNP was found to be 173.91 mg/g. 3.3. Characterization of immobilized ˛-amylase using SEM, EDS, FTIR and XRD The surface characteristics of magnetically immobilized ␣amylase were studied using FE-SEM as shown in Fig. 2. The shape of magnetic nanocomposite of ␣-amylase was confirmed as spherical. The size of the nanocomposite was found to vary from 51.58 nm to
Please cite this article in press as: G. Baskar, et al., Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch, Biochem. Eng. J. (2015), http://dx.doi.org/10.1016/j.bej.2015.02.020
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Fig. 2. SEM analysis of magnetic nanocomposite of ␣-amylase.
21.10 nm and the average particle size was 37.18 nm. The surface of the nanocomposite was found porous in nature which might have enhanced the catalytic activity of nanocomposite of ␣-amylase. Hence the immobilized of ␣-amylase can be recovered from reaction mixture using by the action of magnetic field. The elemental composition of the nanocompoiste was studied using EDS analysis as shown in Fig. 3. The presence of iron, oxygen and carbon was confirmed with the help of EDS analysis. Thus the magnetic effect of nanocomposite of ␣-amylase was confirmed. The functional group present on the surface of nanocomposite of ␣-amylase was analyzed using FTIR as shown in Fig. 4(a). The peaks were observed at 3773.4, 3432.8, 2927.8, 2345.9, 1688.1, 1410.1 and 1101.5. The peak at 3773.4 and 3432.8 represents the presence of N H group with strong primary amine and weak to
moderate secondary amine which confirms the presence of protein (␣-amylase) in magnetic nanocomposite. The peak at 2927.8 represents moderate to strong alkyl bond and peak at 1688.1 represents the C O bond which confirms the presence of aromatic carboxylic acids in nanocomposite. The peak at 1410.1 also represents the presence of weak to strong aromatic C C bond. The presence of alkyl bond and aromatic carboxylic acids might be due to the use of glutaraldehyde as conjugating agent. The results of XRD analysis of magnetic nanocomposite of ␣-amylase is shown in Fig. 4(b). The high peaks were observed at 2 values of 35.56, 57.20 and 62.76. The peak at 35.56 represents diffraction plane (3 1 1), 57.20 corresponds to diffraction plane (5 1 1) and 62.76 corresponds to diffraction plane (4 4 0). Thus the magnetic nanocomposite of ␣amylase was found to be crystalline and cubical in structure. The
Fig. 3. EDS analysis of magnetic nanocomposite of ␣-amylase.
Please cite this article in press as: G. Baskar, et al., Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch, Biochem. Eng. J. (2015), http://dx.doi.org/10.1016/j.bej.2015.02.020
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Fig. 4. (a) FTIR spectrum (b) XRD analysis of magnetic nanocomposite of ␣-amylase.
fungal ␣-amylase from A. oryzae was reported to have three dimensional crystalline structures [16]. Thus the ␣-amylase immobilized on to magnetic nanparticles by simple adsorption, retained its original structure. 3.4. Hydrolysis of sweet potato starch using immobilized ˛-amylase The effect of sweet potato starch concentration on hydrolysis using magnetically immobilized ␣-amylase nanocomposite was observed as shown in Fig. 5(a). The glucose yield was found to increase with increase in starch concentration from 2.5 to 3% (w/v) and reaction time. The decrease in glucose yield was observed for further increase in starch concentration from up to 6%. The amylase activity and percentage conversion of sweet potato starch were found to increase with increases in starch concentration. However the glucose yield was found to decreases with increases in starch concentration. This might be due to the relative decrease in percentage conversion of sweet potato starch into glucose. The maximum glucose yield of 42.56 mg/g was obtained for 3% starch concentration in 80 min of reaction time. The effect of initial pH on hydrolysis of sweet potato starch using magnetically immobilized nanocomposite of ␣-amylase was observed as shown in Fig. 5(b). The initial pH of sweet potato starch solution was varied from 3 to 8 to study its effect on glucose yield. The glucose yield was found to increase with increase in initial pH from 3 to 4 and reaction time. The decrease in glucose yield was observed when the initial pH was further increased from 3 to 8. The maximum glucose yield of 41.34 mg/g was obtained for initial pH 4 in 100 min of reaction time. The effect of reaction temperature on hydrolysis of sweet potato starch was studied by varying the temperature from 25 to 45 ◦ C with an interval of 5 ◦ C as shown in Fig. 5(c). The increase in temperature from 35 to 40 ◦ C was found to increase the glucose yield with increase in reaction time. Then the glucose yield was decreased due
to further increase in temperature to 45 ◦ C. The maximum glucose yield of 41.34 mg/g was obtained in 80 min of reaction time at 40 ◦ C. The effect of varied concentration of nanocomposite of ␣-amylase was studied as shown in Fig. 5(d). The increase in glucose yield was observed for increase in nanocomposite concentration from 0.2 to 1%(w/v). The maximum glucose yield of 41.28 mg/g was obtained in 120 min reaction time using 1% nanocomposite concentration. The immobilized ␣-amylase was recovered using magnetic field and reused for hydrolysis of sweet potato starch (Fig. 6). The immobilized ␣-amylase was found to be active during reuse and 75% its initial hydrolytic activity was obtained even after fifth cycle. 3.5. Kinetics of sweet potato starch hydrolysis by immobilized ˛-amylase The substrate dependency of enzymatic hydrolysis of sweet potato starch was studied in terms of kinetics. The MichaelisMenten kinetics of the enzymatic reactions is given by Eq. (1).
v=
vmax [s] Km + [s]
(1)
where V is the reaction rate, Km is the Michaelis–Menten constant, Vmax is the maximum reaction rate and [S] is the substrate concentration. The Lineweaver–Burk plot is an efficient graphical method of determination of Michaelis–Menten parameters. The Lineweaver–Burk plot provides a more precise way to determine Vmax and Km . The Lineweaver–Burk plot was described by Lineweaver and Burk in 1934 [17,18]. The Michaelis–Menten parameters such as substrate affinity constant (Km ) and substrate saturation constant (Vmax ) obtained from Lineweaver–Burk plot with a correlation co-efficient of 0.9976 (R2 ) were 0.16 mg/ml and 3.63 × 10−3 mol/ml s, respectively. Thus, the magnetically immobilized ␣-amylase has shown high affinity toward sweet potato starch.
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Fig. 5. Optimization of hydrolysis conditions (a) effect of sweet potato starch concentration (♦2.5%; 3.0%; 3.5%; ×4.0%; *4.5%; 䊉5.0%; |5.5%; --6%) (b) Effect of initial pH (♦3; 4; 5; ×6; *7; 䊉8) (c) Effect of reaction temperature (♦25 ◦ C; 30 ◦ C; 35 ◦ C; ×40 ◦ C; *45 ◦ C) (d) effect of magnetically immobilized amylase concentration (♦0.2%; 0.4%; 0.6%; ×0.8%; *1.0%).
in 80 min of reaction time at 40 ◦ C. Thus magnetically immobilized nanocomposite of ␣-amylase can be effectively recovered and reused for maximum utilization thereby decrease in cost production of starch hydrolysate. References
Fig. 6. Relative percentage activity and glucose yield of reused magnetically immobilized ␣-amylase.
4. Conclusions The fungal ␣-amylase enzyme was efficiently immobilized on magnetic nanoparticle and used for hydrolysis of sweet potato starch into glucose. The maximum glucose yield of concentration of 42.89 mg/g was obtained using 3% sweet potato starch with an initial pH of 4 and 1% magnetic nanobiocomposite of ␣-amylase
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Please cite this article in press as: G. Baskar, et al., Magnetic immobilization and characterization of ␣-amylase as nanobiocatalyst for hydrolysis of sweet potato starch, Biochem. Eng. J. (2015), http://dx.doi.org/10.1016/j.bej.2015.02.020