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Immobilization of α-amylase on zirconia: A heterogeneous biocatalyst for starch hydrolysis
Catalysis Communications 8 (2007) 393–399 www.elsevier.com/locate/catcom
Immobilization of a-amylase on zirconia: A heterogeneous biocatalyst for starch hydrolysis R. Reshmi, G. Sanjay, S. Sugunan
*
Department of Applied Chemistry, Cochin University of Science and Technology, Cochin 682 022, Kerala, India Received 17 April 2006; received in revised form 22 June 2006; accepted 10 July 2006 Available online 15 July 2006
Abstract a-Amylase was immobilized on zirconia via adsorption. The support and the immobilized enzymes were characterized using XRD, IR spectra and N2 adsorption studies. The efficiency of immobilized enzymes for starch hydrolysis was tested in a batch reactor. The effect of calcination temperatures on properties of the support as well as upon immobilization was studied. From XRD, IR and N2 adsorption studies it was confirmed that the enzyme was adsorbed on the external surface of the support. pH, buffer concentration and substrate concentration had a significant influence on the activity of immobilized enzyme. Immobilization improved the pH stability of the enzyme. The Michaelis–Menten kinetic constants were calculated from Hanes–Woolf plot. Km for immobilized systems was higher than the free enzyme indicating a decreased affinity by the enzyme for its substrate, which may be due to interparticle diffusional mass transfer restrictions. 2006 Elsevier B.V. All rights reserved. Keywords: a-Amylase; Immobilization; Immobilized enzymes; Zirconia; Adsorption; Starch hydrolysis
1. Introduction Enzymes have been the subject of intense academic interest for many decades and currently poised to become important industrial catalysts. Under the ambit of green chemistry, biocatalysis using renewable resources is very attractive to produce chemicals, which are also safer. Biocatalysis is slowly but steadily gaining importance in various fields of chemical engineering, where chemical synthesis routes are being replaced by enzymatic ones. The major advantage of the enzymatic route is the selectivity with its associated high yield, and exclusivity towards the desired product [1]. The main problems of using the enzymes industrially are the difficulty of their separation from the solution and their inactivation by organic solvent and extreme pH or temperature. Novel designs with immobilized enzymes and without need of separation are of major concern. This also reduces the loss of enzymes and *
Corresponding author. Tel.: +91 484 2575804; fax: +91 484 2577595. E-mail address: [email protected] (S. Sugunan).
1566-7367/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.catcom.2006.07.009
offers the opportunity to use a continuous reactor with a re-use of the enzyme for many reaction cycles and thus lowering the total production cost of enzyme mediated reactions [2]. The immobilized enzyme molecules may also be stabilized against denaturing agents that promote unfolding processes that can destroy the active site [3]. Enhancing both, stabilities can be achieved by immobilization and enzyme engineering (modification of enzyme structure). The traditional acid catalyst methods are now being replaced by enzymic processes [4]. Amylases belong to an enzyme group, which is very commonly used in food and fermentation industry. The hydrolysis of starch to products with low molecular weight, catalyzed by a-amylase (1,4-a-D glucan glucanohydrolase; E.C.3.2.1.1), is one of the most important commercial enzymic processes [5]. Conversion of starch into sugars, syrups and dextrins forms the major part of the starch processing industry. Immobilization of amylase on, mainly, water insoluble carriers, seems to be the most promising way to obtain more stable and reusable forms of enzymes [6]. Immobilization of a-amylase on various particulate
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supports [7], soluble polymers [8] and in ultra filtration membranes [9] has been studied. The disadvantages of polymers as immobilization supports are low pH and thermal stabilities. Porous silica is found to be a good medium for immobilization. Many organic and inorganic supports like clay/modified clays [10], silica [11], zeolite [12] and amorphous aluminium phosphate [13] have been studied for the immobilization of enzymes which are known to be thermally and mechanically stable, non-toxic, and highly resistant against microbial attacks and organic solvents [14]. Solid acid supports can be used to immobilize enzymes since the acidic sites can act as centres of immobilization via the amino groups of enzymes. Immobilization of cellulase, catalase and glucose oxidase on silicate clay minerals has been reported [15–17]. Metal oxides and ceramics were used to immobilize lipase PS (Psuedomonas cepacia) as biocatalyst for the enantioselective acetylation of methyl (±)-mandelate in an ionic liquid solvent system [18]. Increased pH stability and thermal stability was reported for a-amylase immobilized in ordered mesoporous silica (MCM-41, SBA-15 and MCF) [19]. Reports on immobilization of enzymes to metal oxides are limited. As zirconia contains many hydroxyl groups [20] and as the isoelectric point of zirconia (pI 4.8) is near that of free a-amylase (pI 5.8) maximum immobilization takes place at pH 5. Hence they can be used as a carrier for immobilizing a-amylase. The main aim of the present work was to produce an immobilized form of a-amylase with advantageous catalytic properties and stability. The present work deals with the synthesis and characterization of zirconia and its use as support for the immobilization of a-amylase. The effect of the pH on activity as well as stability of the immobilized preparations was studied. In the present study, a-amylase was immobilized on zirconia via adsorption. The effect of different calcination temperatures on the properties of support, before and after immobilization was studied. The activity for starch hydrolysis was evaluated in a batch reactor. The support and the immobilized enzymes were characterized using XRD, IR spectra and N2 adsorption studies. The effect of buffer concentration on the hydrolysis of starch was estimated. The kinetics of the reaction was determined at various substrate concentrations and the kinetic parameters (Km and Vmax) were calculated from the Hanes–Woolf plot. 2. Experimental 2.1. Materials a-Amylase from Bacillus subtilis was procured from Sigma–Aldrich Chemicals Pvt Ltd., Bangalore. Zirconium oxychloride was purchased from CDH Chemicals, Mumbai and ammonium hydroxide used was from Qualigens Fine Chemicals, Mumbai. Starch was obtained from SRL Chemicals, Mumbai. All other chemicals were of highest purity commercially available.
2.2. Preparation of the catalyst Sufficient amount of ammonium hydroxide (1:1 v/v in deionized water) was slowly added to 0.1 M zirconium oxychloride under vigorous stirring at a temperature of 70 C and the pH was adjusted to 10. The precipitate was kept for stirring at this temperature for 12 h and aged for 24 h and then washed with distilled water until free of chloride, filtered, dried in an air oven at 120 C for 12 h and calcined at two different temperatures (500 C and 700 C) for 12 h. 2.3. Immobilization of a-amylase on oxide carriers For adsorption, 1 g of zirconia powder was mixed with equal volumes of 0.1 M phosphate buffer and a-amylase solution. It was shaken in a water bath shaker at required temperature for one and a half hour and then filtered. The filtrate was tested for enzyme protein using the spectrophotometric method of Lowry et al. [21] using Folin Ciocaltaue’s phenol reagent and measuring the absorption at 640 nm in a Shimadzu 160A UV–Vis spectrophotometer [22]. The influence of pH on immobilization was determined by carrying out the immobilization at various pH (4–8) and determining the enzyme activity under constant conditions. The notations of the catalysts are: Z-Zirconia, AZ represent a-amylase adsorbed on zirconia, the numbers 1 and 2 represent calcination temperatures of 500 C and 700 C. 2.4. Characterization of Immobilized enzymes Powder XRD of the immobilized enzyme and the supports were taken on a Rigaku D max-C system with Ni fil˚ ) within the 2h range tered Cu Ka radiation (k 1.5406 A 2–80 at a speed of 2min 1. The samples for XRD were prepared under controlled conditions of humidity. A Micromeritics Gemini 2360 surface area analyzer was used to measure the nitrogen adsorption isotherms of the samples at liquid nitrogen temperature. From this the specific surface area was determined. Prior to the measurement the samples were degassed at room temperature for 12– 16 h in nitrogen flow. The IR spectrum of the samples was obtained using an Nicolet Model Magna IR 560 spectrophotometer using KBr disc method. Changes in the absorption bands were investigated in the 500–4000 cm 1 region. The resolution and acquisition applied were 4 cm 1 and 50 scans respectively. 2.5. Free and immobilized enzyme activity for starch hydrolysis The activities of the free and immobilized enzymes were tested in a batch reactor. 0.1 g immobilized enzyme (1 ml enzyme solution) was mixed with buffered 5% starch solution and shaken in a water bath shaker. 1 ml of the product was mixed with 5 ml iodine solution and the absorbance was read at 610 nm. One unit of enzyme activity is defined
R. Reshmi et al. / Catalysis Communications 8 (2007) 393–399
as the amount required to hydrolyze 1 mg starch per minute under the assay conditions. All results are presented in a normalized form with the activity under optimum conditions being assigned a value of 100%. Influence of pH on activity was determined by carrying out the reaction at varying pH in the range (4–8) and keeping all other reaction conditions constant. The pH stability was determined by taking fixed amount of the catalyst and buffer solution, which was then pre-incubated for different time intervals between 15 min and 20 h at 30 C. The influence of buffer concentration was determined by carrying out the reaction in buffer of concentration range 0.1–0.01 M. The Michaels– Menten kinetics was established from a study of effect of substrate concentration (1–5%) and the Hanes–Woolf plot was employed to calculate Km and Vmax. 3. Results and discussion 3.1. Characterization studies The X-ray diffraction profiles of Z-1 and Z-2 (Fig. 1) were sharp indicating the formation of a crystalline phase. The solids were composed of a mixture of monoclinic, tetragonal and cubic phases which were verified by the means of the presence of peaks at 2h = 28.2 and 31.5 for the monoclinic phase, 30.2 and 35 for the tetragonal phase and 50.5 and 59.5 for the cubic phase [23]. The intense sharp peaks at 27.7 and 31.5 have its maximum intensity expected for the first and second strongest Bragg reflection of the crystalline phase Baddeleyite (Monoclinic ZrO2) [24]. Comparing the two spectrum, a shoulder appeared at 2h = 31.5 in Z-2 indicating that the monoclinic phase was more distinct at higher calcination temperature. The intensities of the peaks corresponding to that of cubic and tetragonal phases decreased in the case of Z-2. In the case of AZ-1, the peak corresponding to 2h = 28.9 and
relative intensity (%)
d
c
b
a 10
20
30
40
50
60
70
80
two theta (degrees) Fig. 1. XRD patterns of support and immobilized a-amylase: (a) Z-1, (b) AZ-1, (c) Z-2 and (d) AZ-2 with enzyme concentration 10 mg/g zirconia.
395
59.9 undergo a shift by 1 and in the case of AZ-2 the distinct planes of monoclinic phase as well as the one at 2h = 35 shifted to lower values. The decrease in peak intensity after enzyme adsorption was more prominent for AZ-2 than AZ-1. The decrease may be due to the strain developed as a result of interaction with enzyme. This finding is in agreement with Harter and Stozky [16] where sorption of catalase on Ca-montmorillonite did not result in expansion of the mineral structure. From the above results it can be concluded that adsorption of a-amylase was entirely external. The spectrum appeared broader after adsorption, which indicates that the enzyme interacts with the support. None of the peaks disappear after adsorption of a-amylase since the concentration of the enzyme taken is only 10 mg/g of the support. The surface area of the metal oxides Z-1, Z-2 and that of the immobilized systems AZ-1, AZ-2 are 91, 36, 79 and 32 m2/g respectively. There was a considerable decrease in the surface area after immobilization. Therefore adsorption of the enzyme was mostly external and slight incorporation of the enzyme occurred within the pores. There was no structure collapse of the support after immobilization because the XRD results indicate a well-ordered structure for AZ-1 and AZ-2. It is proposed that the a-amylase was immobilized either at the external surface or within the pore space near the surface of the support, and thus the immobilized a-amylase on the surface may be more readily available to starch than those present in the pore space. In the IR spectra of Z-1 and Z-2 (Fig. 2), the bands at 3440 cm 1 and 1625 cm 1 are assigned to the bending and stretching vibration of the OH bond in absorbed and coordinated water. Zr–O bond vibrations are known to occur in the frequency range 400–1200 cm 1 [25]. The band at 503 cm 1 resulted from the existence of both tetragonal and monoclinic zirconia. The spectra of ZrOH (Z-1 and Z-2) display the diagnostic absorptions of monoclinic zirconia at 508 cm 1, 580 cm 1 and 730 cm 1 that are associated with the Zr–O bond. The band at 448 cm 1 is the sole IR active Zr–O vibration of cubic ZrO2 [26]. The presence of these phases was also confirmed from the XRD studies. No additional peaks appeared in the case of Z-2. The peak at 448 cm 1 appeared much sharper for Z-2. The OH stretching region undergoes shift to lower values and the bands are broadened after adsorption of a-amylase. Additional peaks due to the groups present in the enzyme appear in the spectra of both AZ-1 and AZ-2. These peaks are due to the CH2 scissoring mode. Hence from the IR studies also it can be concluded that the enzyme was adsorbed at the surface through the OH groups. 3.2. Influence of pH on enzyme activity The pH dependence of the activity of immobilized aamylase has been compared to that of the unmodified enzyme and is presented in Fig. 3a. The maximum activity
R. Reshmi et al. / Catalysis Communications 8 (2007) 393–399
a
100
Activity (%)
396
75
50
25
0 4
5
6
7
8
pH
Activity (%)
b
100 75 50 25 0 0.1
0.05
0.025
0.01
buffer concentration (M) Fig. 3. (a) Influence of pH on activity of: (j) free a-amylase, (m) AZ-1 and (r) AZ-2; reaction conditions: starch concentration – 5%, a-amylase concentration – 10 mg/g zirconia, immobilized enzyme – 0.1 g, buffer concentration – 0.1 M, reactant volume – 20 ml, temperature – 30 C, time – 30 min. (b) Effect of buffer concentration on activity of immobilized a-amylase: (m) AA-1, (r) AA-2; reaction conditions: starch concentration – 5%, a-amylase concentration – 10 mg/g zirconia, immobilized enzyme – 0.1 g, pH 7, reactant volume – 20 ml, temperature – 30 C, time – 30 min.
Fig. 2. IR spectrum of zirconia and immobilized a-amylase: (a) Z-1, (b) AZ-1, (c) Z-2 and (d) AZ-2 with enzyme concentration 10 mg/g zirconia.
of the immobilized enzyme was observed at pH 5. The isoelectric point (pI) of a-amylase is 6, thus at pH 5, the overall met charge of the protein is slightly positive. At this pH, the surface of zirconia has an overall negative charge since the isoelectric point of ZrO2 is pH 4.8. Therefore, an electrostatic interaction between the two is expected. When the immobilization is carried out at a pH less than 5, the same amount of enzyme is immobilized, however the activity of the immobilized enzyme is lower than when the immobilization process is carried out at pH 5. The strength of the electrostatic interaction between the enzyme and the zirconia support is very important in maintaining the overall activity of the enzyme. Free enzyme exhibits maximum activity in the pH range (5–7). At pH 4 and 8 a decrease of the enzymatic activity is observed for both the immobilized and the free enzyme; however, at pH 8
the residual activity of the immobilized enzyme in most samples is significantly higher than that of the unmodified form. Thus, the immobilization process provides a structural stability, preventing an irreversible unfolding of the enzymatic protein. AZ-1 and AZ-2 showed maximum activity in the pH range 6–8. The maximum activity was shown at pH 6 in the case of AZ-1 and AZ-2. The variation of activity with pH, within a range of 2–3 units each side of the pI, is normally a reversible process. The curve profile became much broader between pH 6 and 8, although the curve of free enzyme was peaked at pH 6. The native enzyme could not survive with increase in pH towards alkalinity. This shows that the stability of the enzyme against pH was significantly improved upon immobilization. This expansion is possible due to the stabilization of enzyme molecules resulting from multipoint attachment on the surface of zirconia and due to the charge effects of the support. The immobilized enzyme has same optimum pH as the free enzyme but with a much broader profile [27], which was also beneficial for their applications. The surface of zirconia is covered with –OH groups which are essential to charge neutrality at the surface. On dehydration, OH groups condense to form water leading to the formation of Zr–O sites. As a result of this a slight negative charge may be developed on the surface thereby the pH in
R. Reshmi et al. / Catalysis Communications 8 (2007) 393–399
To determine the effect of buffer concentration on activity, the adsorbed systems were studied at optimum pH and concentration range of 0.01–0.1 M using 5% starch solution (Fig. 3b). In the case of AZ-1 the conversion decreases initially and then remained constant. In the case of AZ-2 there was 100% conversion of starch in the entire concentration range studied. At higher buffer concentration, the ionic strength of the solution is high and therefore it shields the charge effects on the support, thereby diminishing pH differences between the carrier and the bulk solution [31]. As the buffer concentration is lowered, the ionic strength comes down by this means reducing the shielding effect and hence the difference in pH between the carrier and the bulk solution increases bringing down the activity. In the case of oxides calcined at 500 C there is greater number of OH groups and the surface charge developed influence the pH difference between the microenvironment of the enzyme and bulk solution and hence a decrease in activity is observed. At higher calcination temperatures the number of OH groups decreases and hence the charge effects on the support may be reduced so that the increase or decrease in ionic strength has no influence on the pH in the bulk solution and the enzyme microenvironment thereby keeping activity steady.
a
100
Activity(%)
Free a-amylase demonstrates maximum stability at pH 6. Hence the optimum pH of free enzyme was taken as 6. Event though initial activity is high for free amylase (pH 5), stability is lower proving that as time passes by lower pH inactivates the enzyme by changing its conformation. In the active centre protonation is a reversible process, while changes in the charge of structure supporting residues may cause irreversible damage to the native structure. Extremes of pH will however cause a time–dependent, essentially irreversible denaturation. At pH 6 (Fig. 4a) and 7 (Fig. 4b) there is reversible denaturation of the enzyme while at pH 8 (Fig. 4c) there is irreversible denaturation of the enzyme. Hence the optimum pH for free aamylase was taken as 6. Adsorbed enzyme shows increased stability at pH 7 compared to pH 6.This can be taken as evidence for the fact that adsorption causes a shift in pH optimum to the basic side. As time passes, pH causes changes in the protonation at the active centre of the enzyme, which is an irreversible process and hence may cause irreparable damage of the native structure. In the case of AZ-1 and AZ-2 higher stability was obtained at pH 7 even though 100% activity was shown initially at pH 6 and 8. Most of the effects of perturbation of pH
75 50 25 0 0
b Activity(%)
3.3. Effect of buffer concentration on the activity of immobilized a-amylase
3.4. pH stability
15
30
60
120
300
1200
100 75 50 25 0 0
c Activity(%)
the microenvironment of the enzyme will be lower than the bulk pH. A greater bulk pH is required in providing an optimum pH in the microenvironment of the enzyme and hence a shift to higher value is encountered. The immobilized a-amylase displays a greater activity at higher pH values. The pH for maximum activity for immobilized a-amylase shifted to the more alkaline side as compared to the native enzyme, reflecting the amphoteric property of zirconia. The inhibition of activity in the lower pH ranges may be due to two reasons: a lower loading and a possible change of the enzyme conformation due to an unfavorable charge distribution on the amino acid residues [28]. A change in pH will affect the intramolecular hydrogen bonding thus leading to a distorted conformation that will reduce the activity of the enzyme. For amylase immobilization, shifts in the optimum pH towards both the acidic and alkaline directions have been observed [29,22]. The sensitivity to pH is reduced as a result of immobilization [30]. The conformation of the free enzyme will be more favorable in the low pH range so that good activity is obtained. The enzyme is inactivated at lower pH values (pH < 5). In acid solutions (pH < 6), hydrolysis of the labile peptide bonds, sometimes next to aspartic residues may occur. In this case a combination of electrostatic and van der Waal’s interaction might be responsible for the physical adsorption of a-amylase on the support. As the enzymes showed high initial activity at more than one pH, stability measurements had to be performed in order to ascertain the optimum value.
397
15
30
60
120
300
1200
100 75 50 25 0 0
15
60
120
300
1200
Pre-incubation time (min) Fig. 4. Change in activity with respect to pre-incubation time for: (j) free a-amylase, (m) AZ-1 and (r) AZ-2 at pH; (a) 6, (b) 7 and (c) 8; reaction conditions: starch concentration – 5%, a-amylase concentration – 10 mg/g zirconia, immobilized enzyme – 0.1 g, buffer concentration – 0.1 M, reactant volume – 20 ml, temperature – 30 C, reaction time – 30 min.
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optimum observed in immobilized enzyme preparations may be explained by considering the distribution of protons through out the system and analyzing the factors that might lead to relative accumulation or depletion of protons in the microenvironment around the enzyme. The shift towards alkaline region may be explained therefore, by partitioning of protons. When immobilizing an enzyme onto a support, the enzyme often possesses a positive net charge due to the alteration of the proton distribution between the bulk phase and the surroundings of the immobilized enzyme [32]. Negatively charged surface of zirconia will tend to concentrate protons thus lowering pH around the enzyme microenvironment compared to the bulk phase from which the measurements of pH are taken and as a result, immobilized enzyme appears to shift its pH stability profile upwards. The immobilized systems displays significantly improved stability over the free enzyme at higher pH values. Though the pH optimum remained the same for AZ-1 and AZ-2 the maximum stability was shown in the case of AZ-2, which might be due to the increase in crystalline nature at higher temperature and the monoclinic phase being more distinct at higher temperatures. The oxides calcined at 500 C were slightly amorphous in nature and the phases were not as distinct as in the case of oxides calcined at higher temperature. 3.5. Kinetic parameters The kinetic parameters of the adsorbed a-amylase were determined in a batch reactor (Table 1). Km and Vmax are calculated from the Hanes–Woolf plot (Fig. 5). The plot is linear for the free as well as immobilized a-amylase depicting that the enzyme kinetics obeys Michaelis–Menten equation. The Km values for AZ-1 and AZ-2 was approximately 3.8 and 2.8 times higher than that of the free enzyme. Vmax defines the highest possible velocity when all the enzyme is saturated with substrate, therefore, this parameter reflects the intrinsic characteristics of the immobilized enzyme, but may be affected by diffusional constraints. Km is defined as the substrate concentration that gives a reaction velocity of 1/2 Vmax. This parameter reflects the effective characteristics of the enzyme and depends upon both partitioning and diffusional effects. In general, Km of an immobilized enzyme is higher than that of the free enzyme due to diffusional limitations, steric effects and ionic strength [33]. Km shows an increase upon immobilization that reflects in a decreased affinity for the substrate. The change in the affinity for its substrate is also
Table 1 Kinetic parameters for free and immobilized a-amylase Catalyst
Km (·10
Free enzyme AZ-1 AZ-2
2.51 9.53 7.07
4
mol/ml)
Vmax (·10 1.02 0.15 0.86
4
mol/ml/min)
6.45 6.42 6.39 6.36 6.33
S/V
398
(c)
13.4 13.3 13.2 13.1 13.0 12.9
(b)
2.455 2.450 2.445 2.440
(a) 0
2
4
6
8
10 -7
12
14
16
18
20
-1
S (* 10 mol ml ) Fig. 5. Hanes–Woolf plot for: (a) free a-amylase, (b) AZ-1 and (c) AZ-2.
caused by structural changes in the enzyme introduced by the immobilization procedure and lower accessibility of the substrates to the active site of the immobilized enzyme [34]. Similar results were obtained by Moran-Pineda et al. in the case of free, P(HEMA) and P(St-HEMA) bound a-amylase respectively [24]. Bayramoglu et al. [35] have also obtained significantly larger Km and lower Vmax for immobilized a-amylase compared to the free form. Vmax values for immobilized a-amylase on zirconia were lower than that of the free enzyme indicating a lowering of activity of enzyme on account of immobilization. The difference in the Km values in our case was not as apparent as that in other reports. This small increase in Km may be the result of conformational changes and/or diffusional limitations to mass transfer. 4. Conclusions a-Amylase was immobilized on zirconia via adsorption. From XRD and N2 measurement studies it was confirmed that sorption was entirely external. There was a decrease in the intensities of the peaks and a slight broadening after adsorption of a-amylase. The surface area also showed a decrease after adsorption of the enzyme. Additional bands due to the characteristic groups in the enzyme appeared in the IR spectrum of the immobilized systems. Both adsorbed and free enzyme exhibited higher activity at pH 6 but the pH profile was broadened after adsorption. Furthermore, a structural stability of the immobilized enzyme at different pH has been observed. The pH at which the immobilization takes place is very important in maintaining the enzyme function as well as maximizing the amount of enzyme immobilized. It was shown that adsorption of protein on to zirconia requires that the surface charges of support and the protein be compatible. Immobilization improved the pH stability of the enzyme. AZ-1 and AZ-2 exhibited increased stability at pH 7. AZ-2 exhibited greater stability than AZ-1, which might be due to the
R. Reshmi et al. / Catalysis Communications 8 (2007) 393–399
increase in crystalline character at higher temperature. The oxides calcined at 500 C have slightly amorphous nature and the phases were not as distinct as in the case of oxides calcined at higher temperature. An evaluation of the kinetic parameters shows that diffusional limitations to mass transfer is important in batch reactor. The linear nature of Hanes–Woolf plot suggests that immobilized a-amylase obeys Michaelis–Menten kinetics. Km values calculated was higher than the free enzyme, which may be due to the diffusional resistances or conformational changes in the enzyme resulting from immobilization and a lower Vmax values for AZ-1 and AZ-2 than that of the free enzyme was due to lowering of activity of the enzyme on account of immobilization. Acknowledgements The authors thank Sophisticated Instruments Facility, Indian Institute of Science, Bangalore and Sree Chithra Tirunal Institute for Medical Sciences and Technology, Thiruvanthapuram for providing NMR and IR data. References [1] M.D. Lilly, Chem. Eng. Sci. 49 (1994) 151. [2] L.H. Posorske, J. Am. Oil Chem. Soc. 61 (1984) 38. [3] V.V. Mozhaev, Stabilization of proteins by chemical methods, in: W.J.J. Van den Tweel, A. Harder, R.M. Buitelaar (Eds.), Stability and Stabilization of Enzymes, Elsevier, Holland, 1993. [4] F.W. Schenck, R.E. Hebeda, Starch Hydrolysis Products: Worldwide Technology, Production and Applications, VCH, New York, 1992. [5] D. Tanyolac, B.I. Yuruksoy, A.R. Ozdural, J. Biochem. Eng. 2 (1998) 179. [6] A. Kondo, T. Urabe, K. Higashitani, J. Ferment. Bioeng. 77 (1994) 700. [7] M.T. Xavier, V.F. Soares, D.G. Freire, C.P. Moreira, M.F. Mendes, E. Bon, Biomass Bioenerg. 13 (1987) 25. [8] L. Cong, R. Kaul, U. Dissing, B. Mattiasson, J. Biotechnol. 42 (1995) 75. [9] S. Miura, N. Kubota, H. Kawakita, K. Saito, K. Sugita, K. Watanabe, T. Sugo, Radiat. Phys. Chem. 63 (2002) 143.
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Immobilization of a-amylase on zirconia: A heterogeneous biocatalyst for starch hydrolysis R. Reshmi, G. Sanjay, S. Sugunan
*
Department of Applied Chemistry, Cochin University of Science and Technology, Cochin 682 022, Kerala, India Received 17 April 2006; received in revised form 22 June 2006; accepted 10 July 2006 Available online 15 July 2006
Abstract a-Amylase was immobilized on zirconia via adsorption. The support and the immobilized enzymes were characterized using XRD, IR spectra and N2 adsorption studies. The efficiency of immobilized enzymes for starch hydrolysis was tested in a batch reactor. The effect of calcination temperatures on properties of the support as well as upon immobilization was studied. From XRD, IR and N2 adsorption studies it was confirmed that the enzyme was adsorbed on the external surface of the support. pH, buffer concentration and substrate concentration had a significant influence on the activity of immobilized enzyme. Immobilization improved the pH stability of the enzyme. The Michaelis–Menten kinetic constants were calculated from Hanes–Woolf plot. Km for immobilized systems was higher than the free enzyme indicating a decreased affinity by the enzyme for its substrate, which may be due to interparticle diffusional mass transfer restrictions. 2006 Elsevier B.V. All rights reserved. Keywords: a-Amylase; Immobilization; Immobilized enzymes; Zirconia; Adsorption; Starch hydrolysis
1. Introduction Enzymes have been the subject of intense academic interest for many decades and currently poised to become important industrial catalysts. Under the ambit of green chemistry, biocatalysis using renewable resources is very attractive to produce chemicals, which are also safer. Biocatalysis is slowly but steadily gaining importance in various fields of chemical engineering, where chemical synthesis routes are being replaced by enzymatic ones. The major advantage of the enzymatic route is the selectivity with its associated high yield, and exclusivity towards the desired product [1]. The main problems of using the enzymes industrially are the difficulty of their separation from the solution and their inactivation by organic solvent and extreme pH or temperature. Novel designs with immobilized enzymes and without need of separation are of major concern. This also reduces the loss of enzymes and *
Corresponding author. Tel.: +91 484 2575804; fax: +91 484 2577595. E-mail address: [email protected] (S. Sugunan).
1566-7367/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.catcom.2006.07.009
offers the opportunity to use a continuous reactor with a re-use of the enzyme for many reaction cycles and thus lowering the total production cost of enzyme mediated reactions [2]. The immobilized enzyme molecules may also be stabilized against denaturing agents that promote unfolding processes that can destroy the active site [3]. Enhancing both, stabilities can be achieved by immobilization and enzyme engineering (modification of enzyme structure). The traditional acid catalyst methods are now being replaced by enzymic processes [4]. Amylases belong to an enzyme group, which is very commonly used in food and fermentation industry. The hydrolysis of starch to products with low molecular weight, catalyzed by a-amylase (1,4-a-D glucan glucanohydrolase; E.C.3.2.1.1), is one of the most important commercial enzymic processes [5]. Conversion of starch into sugars, syrups and dextrins forms the major part of the starch processing industry. Immobilization of amylase on, mainly, water insoluble carriers, seems to be the most promising way to obtain more stable and reusable forms of enzymes [6]. Immobilization of a-amylase on various particulate
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supports [7], soluble polymers [8] and in ultra filtration membranes [9] has been studied. The disadvantages of polymers as immobilization supports are low pH and thermal stabilities. Porous silica is found to be a good medium for immobilization. Many organic and inorganic supports like clay/modified clays [10], silica [11], zeolite [12] and amorphous aluminium phosphate [13] have been studied for the immobilization of enzymes which are known to be thermally and mechanically stable, non-toxic, and highly resistant against microbial attacks and organic solvents [14]. Solid acid supports can be used to immobilize enzymes since the acidic sites can act as centres of immobilization via the amino groups of enzymes. Immobilization of cellulase, catalase and glucose oxidase on silicate clay minerals has been reported [15–17]. Metal oxides and ceramics were used to immobilize lipase PS (Psuedomonas cepacia) as biocatalyst for the enantioselective acetylation of methyl (±)-mandelate in an ionic liquid solvent system [18]. Increased pH stability and thermal stability was reported for a-amylase immobilized in ordered mesoporous silica (MCM-41, SBA-15 and MCF) [19]. Reports on immobilization of enzymes to metal oxides are limited. As zirconia contains many hydroxyl groups [20] and as the isoelectric point of zirconia (pI 4.8) is near that of free a-amylase (pI 5.8) maximum immobilization takes place at pH 5. Hence they can be used as a carrier for immobilizing a-amylase. The main aim of the present work was to produce an immobilized form of a-amylase with advantageous catalytic properties and stability. The present work deals with the synthesis and characterization of zirconia and its use as support for the immobilization of a-amylase. The effect of the pH on activity as well as stability of the immobilized preparations was studied. In the present study, a-amylase was immobilized on zirconia via adsorption. The effect of different calcination temperatures on the properties of support, before and after immobilization was studied. The activity for starch hydrolysis was evaluated in a batch reactor. The support and the immobilized enzymes were characterized using XRD, IR spectra and N2 adsorption studies. The effect of buffer concentration on the hydrolysis of starch was estimated. The kinetics of the reaction was determined at various substrate concentrations and the kinetic parameters (Km and Vmax) were calculated from the Hanes–Woolf plot. 2. Experimental 2.1. Materials a-Amylase from Bacillus subtilis was procured from Sigma–Aldrich Chemicals Pvt Ltd., Bangalore. Zirconium oxychloride was purchased from CDH Chemicals, Mumbai and ammonium hydroxide used was from Qualigens Fine Chemicals, Mumbai. Starch was obtained from SRL Chemicals, Mumbai. All other chemicals were of highest purity commercially available.
2.2. Preparation of the catalyst Sufficient amount of ammonium hydroxide (1:1 v/v in deionized water) was slowly added to 0.1 M zirconium oxychloride under vigorous stirring at a temperature of 70 C and the pH was adjusted to 10. The precipitate was kept for stirring at this temperature for 12 h and aged for 24 h and then washed with distilled water until free of chloride, filtered, dried in an air oven at 120 C for 12 h and calcined at two different temperatures (500 C and 700 C) for 12 h. 2.3. Immobilization of a-amylase on oxide carriers For adsorption, 1 g of zirconia powder was mixed with equal volumes of 0.1 M phosphate buffer and a-amylase solution. It was shaken in a water bath shaker at required temperature for one and a half hour and then filtered. The filtrate was tested for enzyme protein using the spectrophotometric method of Lowry et al. [21] using Folin Ciocaltaue’s phenol reagent and measuring the absorption at 640 nm in a Shimadzu 160A UV–Vis spectrophotometer [22]. The influence of pH on immobilization was determined by carrying out the immobilization at various pH (4–8) and determining the enzyme activity under constant conditions. The notations of the catalysts are: Z-Zirconia, AZ represent a-amylase adsorbed on zirconia, the numbers 1 and 2 represent calcination temperatures of 500 C and 700 C. 2.4. Characterization of Immobilized enzymes Powder XRD of the immobilized enzyme and the supports were taken on a Rigaku D max-C system with Ni fil˚ ) within the 2h range tered Cu Ka radiation (k 1.5406 A 2–80 at a speed of 2min 1. The samples for XRD were prepared under controlled conditions of humidity. A Micromeritics Gemini 2360 surface area analyzer was used to measure the nitrogen adsorption isotherms of the samples at liquid nitrogen temperature. From this the specific surface area was determined. Prior to the measurement the samples were degassed at room temperature for 12– 16 h in nitrogen flow. The IR spectrum of the samples was obtained using an Nicolet Model Magna IR 560 spectrophotometer using KBr disc method. Changes in the absorption bands were investigated in the 500–4000 cm 1 region. The resolution and acquisition applied were 4 cm 1 and 50 scans respectively. 2.5. Free and immobilized enzyme activity for starch hydrolysis The activities of the free and immobilized enzymes were tested in a batch reactor. 0.1 g immobilized enzyme (1 ml enzyme solution) was mixed with buffered 5% starch solution and shaken in a water bath shaker. 1 ml of the product was mixed with 5 ml iodine solution and the absorbance was read at 610 nm. One unit of enzyme activity is defined
R. Reshmi et al. / Catalysis Communications 8 (2007) 393–399
as the amount required to hydrolyze 1 mg starch per minute under the assay conditions. All results are presented in a normalized form with the activity under optimum conditions being assigned a value of 100%. Influence of pH on activity was determined by carrying out the reaction at varying pH in the range (4–8) and keeping all other reaction conditions constant. The pH stability was determined by taking fixed amount of the catalyst and buffer solution, which was then pre-incubated for different time intervals between 15 min and 20 h at 30 C. The influence of buffer concentration was determined by carrying out the reaction in buffer of concentration range 0.1–0.01 M. The Michaels– Menten kinetics was established from a study of effect of substrate concentration (1–5%) and the Hanes–Woolf plot was employed to calculate Km and Vmax. 3. Results and discussion 3.1. Characterization studies The X-ray diffraction profiles of Z-1 and Z-2 (Fig. 1) were sharp indicating the formation of a crystalline phase. The solids were composed of a mixture of monoclinic, tetragonal and cubic phases which were verified by the means of the presence of peaks at 2h = 28.2 and 31.5 for the monoclinic phase, 30.2 and 35 for the tetragonal phase and 50.5 and 59.5 for the cubic phase [23]. The intense sharp peaks at 27.7 and 31.5 have its maximum intensity expected for the first and second strongest Bragg reflection of the crystalline phase Baddeleyite (Monoclinic ZrO2) [24]. Comparing the two spectrum, a shoulder appeared at 2h = 31.5 in Z-2 indicating that the monoclinic phase was more distinct at higher calcination temperature. The intensities of the peaks corresponding to that of cubic and tetragonal phases decreased in the case of Z-2. In the case of AZ-1, the peak corresponding to 2h = 28.9 and
relative intensity (%)
d
c
b
a 10
20
30
40
50
60
70
80
two theta (degrees) Fig. 1. XRD patterns of support and immobilized a-amylase: (a) Z-1, (b) AZ-1, (c) Z-2 and (d) AZ-2 with enzyme concentration 10 mg/g zirconia.
395
59.9 undergo a shift by 1 and in the case of AZ-2 the distinct planes of monoclinic phase as well as the one at 2h = 35 shifted to lower values. The decrease in peak intensity after enzyme adsorption was more prominent for AZ-2 than AZ-1. The decrease may be due to the strain developed as a result of interaction with enzyme. This finding is in agreement with Harter and Stozky [16] where sorption of catalase on Ca-montmorillonite did not result in expansion of the mineral structure. From the above results it can be concluded that adsorption of a-amylase was entirely external. The spectrum appeared broader after adsorption, which indicates that the enzyme interacts with the support. None of the peaks disappear after adsorption of a-amylase since the concentration of the enzyme taken is only 10 mg/g of the support. The surface area of the metal oxides Z-1, Z-2 and that of the immobilized systems AZ-1, AZ-2 are 91, 36, 79 and 32 m2/g respectively. There was a considerable decrease in the surface area after immobilization. Therefore adsorption of the enzyme was mostly external and slight incorporation of the enzyme occurred within the pores. There was no structure collapse of the support after immobilization because the XRD results indicate a well-ordered structure for AZ-1 and AZ-2. It is proposed that the a-amylase was immobilized either at the external surface or within the pore space near the surface of the support, and thus the immobilized a-amylase on the surface may be more readily available to starch than those present in the pore space. In the IR spectra of Z-1 and Z-2 (Fig. 2), the bands at 3440 cm 1 and 1625 cm 1 are assigned to the bending and stretching vibration of the OH bond in absorbed and coordinated water. Zr–O bond vibrations are known to occur in the frequency range 400–1200 cm 1 [25]. The band at 503 cm 1 resulted from the existence of both tetragonal and monoclinic zirconia. The spectra of ZrOH (Z-1 and Z-2) display the diagnostic absorptions of monoclinic zirconia at 508 cm 1, 580 cm 1 and 730 cm 1 that are associated with the Zr–O bond. The band at 448 cm 1 is the sole IR active Zr–O vibration of cubic ZrO2 [26]. The presence of these phases was also confirmed from the XRD studies. No additional peaks appeared in the case of Z-2. The peak at 448 cm 1 appeared much sharper for Z-2. The OH stretching region undergoes shift to lower values and the bands are broadened after adsorption of a-amylase. Additional peaks due to the groups present in the enzyme appear in the spectra of both AZ-1 and AZ-2. These peaks are due to the CH2 scissoring mode. Hence from the IR studies also it can be concluded that the enzyme was adsorbed at the surface through the OH groups. 3.2. Influence of pH on enzyme activity The pH dependence of the activity of immobilized aamylase has been compared to that of the unmodified enzyme and is presented in Fig. 3a. The maximum activity
R. Reshmi et al. / Catalysis Communications 8 (2007) 393–399
a
100
Activity (%)
396
75
50
25
0 4
5
6
7
8
pH
Activity (%)
b
100 75 50 25 0 0.1
0.05
0.025
0.01
buffer concentration (M) Fig. 3. (a) Influence of pH on activity of: (j) free a-amylase, (m) AZ-1 and (r) AZ-2; reaction conditions: starch concentration – 5%, a-amylase concentration – 10 mg/g zirconia, immobilized enzyme – 0.1 g, buffer concentration – 0.1 M, reactant volume – 20 ml, temperature – 30 C, time – 30 min. (b) Effect of buffer concentration on activity of immobilized a-amylase: (m) AA-1, (r) AA-2; reaction conditions: starch concentration – 5%, a-amylase concentration – 10 mg/g zirconia, immobilized enzyme – 0.1 g, pH 7, reactant volume – 20 ml, temperature – 30 C, time – 30 min.
Fig. 2. IR spectrum of zirconia and immobilized a-amylase: (a) Z-1, (b) AZ-1, (c) Z-2 and (d) AZ-2 with enzyme concentration 10 mg/g zirconia.
of the immobilized enzyme was observed at pH 5. The isoelectric point (pI) of a-amylase is 6, thus at pH 5, the overall met charge of the protein is slightly positive. At this pH, the surface of zirconia has an overall negative charge since the isoelectric point of ZrO2 is pH 4.8. Therefore, an electrostatic interaction between the two is expected. When the immobilization is carried out at a pH less than 5, the same amount of enzyme is immobilized, however the activity of the immobilized enzyme is lower than when the immobilization process is carried out at pH 5. The strength of the electrostatic interaction between the enzyme and the zirconia support is very important in maintaining the overall activity of the enzyme. Free enzyme exhibits maximum activity in the pH range (5–7). At pH 4 and 8 a decrease of the enzymatic activity is observed for both the immobilized and the free enzyme; however, at pH 8
the residual activity of the immobilized enzyme in most samples is significantly higher than that of the unmodified form. Thus, the immobilization process provides a structural stability, preventing an irreversible unfolding of the enzymatic protein. AZ-1 and AZ-2 showed maximum activity in the pH range 6–8. The maximum activity was shown at pH 6 in the case of AZ-1 and AZ-2. The variation of activity with pH, within a range of 2–3 units each side of the pI, is normally a reversible process. The curve profile became much broader between pH 6 and 8, although the curve of free enzyme was peaked at pH 6. The native enzyme could not survive with increase in pH towards alkalinity. This shows that the stability of the enzyme against pH was significantly improved upon immobilization. This expansion is possible due to the stabilization of enzyme molecules resulting from multipoint attachment on the surface of zirconia and due to the charge effects of the support. The immobilized enzyme has same optimum pH as the free enzyme but with a much broader profile [27], which was also beneficial for their applications. The surface of zirconia is covered with –OH groups which are essential to charge neutrality at the surface. On dehydration, OH groups condense to form water leading to the formation of Zr–O sites. As a result of this a slight negative charge may be developed on the surface thereby the pH in
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To determine the effect of buffer concentration on activity, the adsorbed systems were studied at optimum pH and concentration range of 0.01–0.1 M using 5% starch solution (Fig. 3b). In the case of AZ-1 the conversion decreases initially and then remained constant. In the case of AZ-2 there was 100% conversion of starch in the entire concentration range studied. At higher buffer concentration, the ionic strength of the solution is high and therefore it shields the charge effects on the support, thereby diminishing pH differences between the carrier and the bulk solution [31]. As the buffer concentration is lowered, the ionic strength comes down by this means reducing the shielding effect and hence the difference in pH between the carrier and the bulk solution increases bringing down the activity. In the case of oxides calcined at 500 C there is greater number of OH groups and the surface charge developed influence the pH difference between the microenvironment of the enzyme and bulk solution and hence a decrease in activity is observed. At higher calcination temperatures the number of OH groups decreases and hence the charge effects on the support may be reduced so that the increase or decrease in ionic strength has no influence on the pH in the bulk solution and the enzyme microenvironment thereby keeping activity steady.
a
100
Activity(%)
Free a-amylase demonstrates maximum stability at pH 6. Hence the optimum pH of free enzyme was taken as 6. Event though initial activity is high for free amylase (pH 5), stability is lower proving that as time passes by lower pH inactivates the enzyme by changing its conformation. In the active centre protonation is a reversible process, while changes in the charge of structure supporting residues may cause irreversible damage to the native structure. Extremes of pH will however cause a time–dependent, essentially irreversible denaturation. At pH 6 (Fig. 4a) and 7 (Fig. 4b) there is reversible denaturation of the enzyme while at pH 8 (Fig. 4c) there is irreversible denaturation of the enzyme. Hence the optimum pH for free aamylase was taken as 6. Adsorbed enzyme shows increased stability at pH 7 compared to pH 6.This can be taken as evidence for the fact that adsorption causes a shift in pH optimum to the basic side. As time passes, pH causes changes in the protonation at the active centre of the enzyme, which is an irreversible process and hence may cause irreparable damage of the native structure. In the case of AZ-1 and AZ-2 higher stability was obtained at pH 7 even though 100% activity was shown initially at pH 6 and 8. Most of the effects of perturbation of pH
75 50 25 0 0
b Activity(%)
3.3. Effect of buffer concentration on the activity of immobilized a-amylase
3.4. pH stability
15
30
60
120
300
1200
100 75 50 25 0 0
c Activity(%)
the microenvironment of the enzyme will be lower than the bulk pH. A greater bulk pH is required in providing an optimum pH in the microenvironment of the enzyme and hence a shift to higher value is encountered. The immobilized a-amylase displays a greater activity at higher pH values. The pH for maximum activity for immobilized a-amylase shifted to the more alkaline side as compared to the native enzyme, reflecting the amphoteric property of zirconia. The inhibition of activity in the lower pH ranges may be due to two reasons: a lower loading and a possible change of the enzyme conformation due to an unfavorable charge distribution on the amino acid residues [28]. A change in pH will affect the intramolecular hydrogen bonding thus leading to a distorted conformation that will reduce the activity of the enzyme. For amylase immobilization, shifts in the optimum pH towards both the acidic and alkaline directions have been observed [29,22]. The sensitivity to pH is reduced as a result of immobilization [30]. The conformation of the free enzyme will be more favorable in the low pH range so that good activity is obtained. The enzyme is inactivated at lower pH values (pH < 5). In acid solutions (pH < 6), hydrolysis of the labile peptide bonds, sometimes next to aspartic residues may occur. In this case a combination of electrostatic and van der Waal’s interaction might be responsible for the physical adsorption of a-amylase on the support. As the enzymes showed high initial activity at more than one pH, stability measurements had to be performed in order to ascertain the optimum value.
397
15
30
60
120
300
1200
100 75 50 25 0 0
15
60
120
300
1200
Pre-incubation time (min) Fig. 4. Change in activity with respect to pre-incubation time for: (j) free a-amylase, (m) AZ-1 and (r) AZ-2 at pH; (a) 6, (b) 7 and (c) 8; reaction conditions: starch concentration – 5%, a-amylase concentration – 10 mg/g zirconia, immobilized enzyme – 0.1 g, buffer concentration – 0.1 M, reactant volume – 20 ml, temperature – 30 C, reaction time – 30 min.
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optimum observed in immobilized enzyme preparations may be explained by considering the distribution of protons through out the system and analyzing the factors that might lead to relative accumulation or depletion of protons in the microenvironment around the enzyme. The shift towards alkaline region may be explained therefore, by partitioning of protons. When immobilizing an enzyme onto a support, the enzyme often possesses a positive net charge due to the alteration of the proton distribution between the bulk phase and the surroundings of the immobilized enzyme [32]. Negatively charged surface of zirconia will tend to concentrate protons thus lowering pH around the enzyme microenvironment compared to the bulk phase from which the measurements of pH are taken and as a result, immobilized enzyme appears to shift its pH stability profile upwards. The immobilized systems displays significantly improved stability over the free enzyme at higher pH values. Though the pH optimum remained the same for AZ-1 and AZ-2 the maximum stability was shown in the case of AZ-2, which might be due to the increase in crystalline nature at higher temperature and the monoclinic phase being more distinct at higher temperatures. The oxides calcined at 500 C were slightly amorphous in nature and the phases were not as distinct as in the case of oxides calcined at higher temperature. 3.5. Kinetic parameters The kinetic parameters of the adsorbed a-amylase were determined in a batch reactor (Table 1). Km and Vmax are calculated from the Hanes–Woolf plot (Fig. 5). The plot is linear for the free as well as immobilized a-amylase depicting that the enzyme kinetics obeys Michaelis–Menten equation. The Km values for AZ-1 and AZ-2 was approximately 3.8 and 2.8 times higher than that of the free enzyme. Vmax defines the highest possible velocity when all the enzyme is saturated with substrate, therefore, this parameter reflects the intrinsic characteristics of the immobilized enzyme, but may be affected by diffusional constraints. Km is defined as the substrate concentration that gives a reaction velocity of 1/2 Vmax. This parameter reflects the effective characteristics of the enzyme and depends upon both partitioning and diffusional effects. In general, Km of an immobilized enzyme is higher than that of the free enzyme due to diffusional limitations, steric effects and ionic strength [33]. Km shows an increase upon immobilization that reflects in a decreased affinity for the substrate. The change in the affinity for its substrate is also
Table 1 Kinetic parameters for free and immobilized a-amylase Catalyst
Km (·10
Free enzyme AZ-1 AZ-2
2.51 9.53 7.07
4
mol/ml)
Vmax (·10 1.02 0.15 0.86
4
mol/ml/min)
6.45 6.42 6.39 6.36 6.33
S/V
398
(c)
13.4 13.3 13.2 13.1 13.0 12.9
(b)
2.455 2.450 2.445 2.440
(a) 0
2
4
6
8
10 -7
12
14
16
18
20
-1
S (* 10 mol ml ) Fig. 5. Hanes–Woolf plot for: (a) free a-amylase, (b) AZ-1 and (c) AZ-2.
caused by structural changes in the enzyme introduced by the immobilization procedure and lower accessibility of the substrates to the active site of the immobilized enzyme [34]. Similar results were obtained by Moran-Pineda et al. in the case of free, P(HEMA) and P(St-HEMA) bound a-amylase respectively [24]. Bayramoglu et al. [35] have also obtained significantly larger Km and lower Vmax for immobilized a-amylase compared to the free form. Vmax values for immobilized a-amylase on zirconia were lower than that of the free enzyme indicating a lowering of activity of enzyme on account of immobilization. The difference in the Km values in our case was not as apparent as that in other reports. This small increase in Km may be the result of conformational changes and/or diffusional limitations to mass transfer. 4. Conclusions a-Amylase was immobilized on zirconia via adsorption. From XRD and N2 measurement studies it was confirmed that sorption was entirely external. There was a decrease in the intensities of the peaks and a slight broadening after adsorption of a-amylase. The surface area also showed a decrease after adsorption of the enzyme. Additional bands due to the characteristic groups in the enzyme appeared in the IR spectrum of the immobilized systems. Both adsorbed and free enzyme exhibited higher activity at pH 6 but the pH profile was broadened after adsorption. Furthermore, a structural stability of the immobilized enzyme at different pH has been observed. The pH at which the immobilization takes place is very important in maintaining the enzyme function as well as maximizing the amount of enzyme immobilized. It was shown that adsorption of protein on to zirconia requires that the surface charges of support and the protein be compatible. Immobilization improved the pH stability of the enzyme. AZ-1 and AZ-2 exhibited increased stability at pH 7. AZ-2 exhibited greater stability than AZ-1, which might be due to the
R. Reshmi et al. / Catalysis Communications 8 (2007) 393–399
increase in crystalline character at higher temperature. The oxides calcined at 500 C have slightly amorphous nature and the phases were not as distinct as in the case of oxides calcined at higher temperature. An evaluation of the kinetic parameters shows that diffusional limitations to mass transfer is important in batch reactor. The linear nature of Hanes–Woolf plot suggests that immobilized a-amylase obeys Michaelis–Menten kinetics. Km values calculated was higher than the free enzyme, which may be due to the diffusional resistances or conformational changes in the enzyme resulting from immobilization and a lower Vmax values for AZ-1 and AZ-2 than that of the free enzyme was due to lowering of activity of the enzyme on account of immobilization. Acknowledgements The authors thank Sophisticated Instruments Facility, Indian Institute of Science, Bangalore and Sree Chithra Tirunal Institute for Medical Sciences and Technology, Thiruvanthapuram for providing NMR and IR data. References [1] M.D. Lilly, Chem. Eng. Sci. 49 (1994) 151. [2] L.H. Posorske, J. Am. Oil Chem. Soc. 61 (1984) 38. [3] V.V. Mozhaev, Stabilization of proteins by chemical methods, in: W.J.J. Van den Tweel, A. Harder, R.M. Buitelaar (Eds.), Stability and Stabilization of Enzymes, Elsevier, Holland, 1993. [4] F.W. Schenck, R.E. Hebeda, Starch Hydrolysis Products: Worldwide Technology, Production and Applications, VCH, New York, 1992. [5] D. Tanyolac, B.I. Yuruksoy, A.R. Ozdural, J. Biochem. Eng. 2 (1998) 179. [6] A. Kondo, T. Urabe, K. Higashitani, J. Ferment. Bioeng. 77 (1994) 700. [7] M.T. Xavier, V.F. Soares, D.G. Freire, C.P. Moreira, M.F. Mendes, E. Bon, Biomass Bioenerg. 13 (1987) 25. [8] L. Cong, R. Kaul, U. Dissing, B. Mattiasson, J. Biotechnol. 42 (1995) 75. [9] S. Miura, N. Kubota, H. Kawakita, K. Saito, K. Sugita, K. Watanabe, T. Sugo, Radiat. Phys. Chem. 63 (2002) 143.
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