- Email: [email protected]
Bentonite and sepiolite as supporting media: Immobilization of catalase
Applied Clay Science 65–66 (2012) 114–120
Contents lists available at SciVerse ScienceDirect
Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Research paper
Bentonite and sepiolite as supporting media: Immobilization of catalase Sevilay Cengiz a, Levent Çavaş b, Kadir Yurdakoç b,⁎ a b
Akdeniz University, Faculty of Arts and Sciences, Department of Chemistry, 07058 Antalya, Turkey Dokuz Eylul University, Faculty of Science, Department of Chemistry, Tinaztepe Campus, 35160 Buca/Izmir, Turkey
a r t i c l e
i n f o
Article history: Received 9 June 2011 Received in revised form 4 June 2012 Accepted 5 June 2012 Available online 21 July 2012 Keywords: Bentonite Sepiolite Clay Adsorption Immobilization Catalase
a b s t r a c t Immobilization of catalase onto bentonite and sepiolite was studied as a function of pH, temperature and ionic strength for free and immobilized enzyme activities. The optimum temperature, pH and ionic strength for free and immobilized catalases were 35 °C, pH=7 and 50 mM (for free catalase) and 35 °C, pH=9 and 50 mM (for both immobilized catalases), respectively. Km and Vmax values were 116.8 mM and 30.1 μmol min−1 mg protein− 1 for free catalase, 55.6 mM, Vmax =6.6 μmol min−1 mg protein− 1 for bentonite supported catalase and Km = 49.2 mM, Vmax = 8.7 μmol min− 1 mg protein− 1 for sepiolite supported catalase. The storage stabilities of immobilized catalase were higher than free catalase. Free catalase lost 50.7% of its initial activity, while sepiolite immobilized catalase lost 41.1% and bentonite immobilized catalase lost only 32.6% of their initial activities for 1 h preincubation time at 45 °C. Both immobilized catalases showed an operational stability and for a period of 10 cycles of experiments they lost only 34.4% and 9.8% of their initial activities for bentonite and sepiolite immobilized catalases, respectively. Batch adsorption studies were also carried out to determine the kinetic parameters. The pseudo second order model provided the best correlation with the experimental results. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Enzymes have useful applications in various industrial fields such as biotechnological, pharmaceutical and food industries (Li et al., 2004). Enzymes have taken lots of interests from a lot of researchers owing to unique properties such as catalytic efficiency, specificity and mild conditions of operation. Although these properties enable enzymes to catalyze a series of biotransformation reactions, their usage is restricted with some problems including reusing of free enzymes, limited stability against high temperatures, pHs, ionic strengths and inhibitory sensitivities (Krajewska, 2009). There are some methods to cope with these problems. One of them is immobilization that can be made by some techniques such as adsorption, covalent binding, and entrapment vb. Among these, adsorption of enzymes onto solid supports is preferred in order to enhance the industrial utility, to improve their stability and to provide their continuous use (Cabana et al., 2009). The basic requirements of support materials which are used for enzyme immobilization are mechanical and thermal strength, microbial resistance, and non-toxicity, and regenerability, low cost and also large surface area for enzymic reactions (Kilara, 1981; Li et al., 2004). Up to now, various types of support materials such as chitosan (Shentu et al., 2005), a crosslinked macromolecular carrier of a polysaccharide structure (gellan) (Popa et al., 2006), chitosan–clay composite (Chang and Juang, 2007), Cu(II) adsorbed
⁎ Corresponding author. Tel.: + 90 232 3018695; fax: + 90 232 4534188. E-mail address: [email protected] (K. Yurdakoç). 0169-1317/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2012.06.004
chitosan beads (Cetinus et al., 2009), microporous polyamide hollowfibers (Akgöl et al., 2003), a novel bentonite–cysteine (Bent–Cys) microcomposite (Öztürk et al., 2008), magnesium silicate (Tukel and Alptekin, 2004), eggshell membrane (Choi and Yiu, 2004), alumina and cellulose (Eberhardt et al., 2004), crosslinked polystyrene ethylene glycol acrylate resin (CLPSER) (Kumar and Kumar, 2005), tetraethoxyorthosilicate (TEOS) based sol–gels (Domink et al., 2006), surface-modified carbon blacks (Caillou et al., 2008), cotton fabric (Wang et al., 2008), mesoporous silica (Itoh et al., 2009a), silica–alumina inorganic composite membrane (Itoh et al., 2009b), bentonite (Alkan et al., 2005), and modified perlite (Tarhan and Telefoncu, 1992) have been used for catalase immobilization. Immobilization of alkaline phosphatase on Na-sepiolite and modified sepiolite was studied in details (Carrasco et al., 1995; Sedaghat et al., 2009). Adsorption and electrokinetic properties of casein and catalase onto sepiolite were also investigated (Demirbas and Alkan, 2011). However, adsorption of catalase onto sepiolite, or usage of sepiolite for immobilization of catalase was still not studied so much, because there is very limited information in the literature. In the light of the knowledge mentioned above bentonite and especially sepiolite were chosen in this study as a support because of their low cost, high specific surface area, chemical, mechanical and thermal stability, porous structure and high adsorption capacities. Catalase (EC 1.11.1.6) that consists of four protein subunits is an abundant native enzyme, which decomposes H2O2 to water and oxygen (Aebi, 1974). Immobilized catalase is used in many industrial processes such as textile (Costa et al., 2002; Eberhardt et al., 2004)
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
and food industries (Akgöl et al., 2001; Kubal and D'Souza, 2004), treatment of waste waters and also in contact-lenses solutions, cosmetics and pharmaceutical formulations (Choi and Yiu, 2004) to determine the quantity of H2O2 or to remove the excess of H2O2 (Tarhan, 1995). Therefore, the present study aimed to investigate the immobilization characteristics of catalase onto bentonite and sepiolite from Turkey. Besides the kinetic parameters, thermal, storage and operational stabilities of free and immobilized catalases will also be determined and compared to each other.
115
by catalase at 240 nm. The amount of H2O2 converted into H2O and O2 in 1 min under standard conditions is accepted as the enzyme reaction rate. The optimum temperature, pH and ionic strength values were determined for free and immobilized enzymes. Michaelis constants (Km) and maximum rate (Vmax) values for free and immobilized catalases were also determined from the slope of the x-axis and the intercept of the y-axis, respectively according to below equation (Atkins and Paula, 2006). 1 1 KM 1 ¼ þ : ⋅ V V max V max ½S0
ð1Þ
2. Materials and methods 2.5. Determination of thermal stability of catalase
2.1. Clays and catalase Bentonite, a sodium–calcium-rich smectite (NaCaS) of light yellowish green color and sepiolite (S) were obtained from Enez/Edirne and Eskisehir-Turkey, respectively and used as supporting agents in the experiments. The properties of the clays were summarized in Table 1. Catalase was obtained from Sigma (Sigma Cat. No. C 9322) and used without further purification and treatment. In the preparation of catalase solution, phosphate buffer (pH = 7, 50 mM) was used. 2.2. XRD analysis X-ray diffraction patterns of the clays (XRD) were obtained with oriented mounts, in a Phillips X'Pert Pro instrument using Cu Kα radiation with a wavelength of 1.5418 Å, and range for bentonite 2θ = 3–10° and 3–70°, for sepiolite 2θ = 5–30°. 2.3. Immobilization of catalase onto clays 5 mg of bentonite or sepiolite was added into 1 mL catalase solutions that have varying concentrations between 30 and 200 μg mL− 1. The samples were agitated at 105 rpm until these reach the equilibrium conditions in a temperature controlled shaker (Memmert). The immobilized catalase was separated from the residual aliquot by centrifugation at 5000 rpm for 5 min at +4 °C (Hettich, R21). The amount of catalase immobilized onto clays was calculated by measuring the initial and equilibrium concentrations of catalase in the medium by the method of Bradford (1976). The pH of the immobilization medium was varied from 3 to 12 by using different buffer systems (50 mM CH3COOH for pH =3–5; 50 mM KH2PO4–K2HPO4 for pH = 7–9 and 50 mM NaOH– KCl for pH = 12). The temperature and the ionic strength of the immobilization medium were varied from 15 °C to 65 °C and from 0.01 to 1 M, respectively. 2.4. Catalase activity assay The activity of free and immobilized catalases was determined spectrophotometrically (Shimadzu UV–Visible 1601) at 240 nm with Aebi method (Aebi, 1974). This method is based on the principle that the absorbance will decrease due to the decomposition of hydrogen peroxide
Table 1 Chemical composition of bentonite and sepiolite (%). Component
Bentonite
Sepiolite
SiO2 Al2O3 Fe2O3 MgO CaO Na2O K2O Sulfate Ignition loss
57.8 19.2 3.6 2.1 4.2 2.7 1.4 – 7.5
48.0 0.5 0.4 24.0 4.0 0.02 0.03 0.02 23.0
The effect of temperature on the activities of free and immobilized catalases was determined at 35 °C, thereafter incubating the samples in phosphate buffers (pH = 7) at various temperatures (15–65 °C) for specified times. 2.6. Determination of storage stability of catalase In order to find out the storage stabilities of free and immobilized catalases at various temperatures (−18 °C, 4 °C and 25 °C), the samples were incubated in phosphate buffer (pH = 7) for days and their activities were found periodically at 35 °C. 2.7. Determination of operational stability of catalase To identify the operational stabilities of immobilized catalase, prepared samples were added into substrate solution and stirred slowly. Then the solution was separated from immobilized enzyme and its absorbance was measured at 240 nm. Immobilized catalase was washed with phosphate buffer (pH = 7), then the same experiments were repeated several times. 2.8. Immobilization kinetics The immobilization experiments were done with 5 mg of the clays at 35 °C. The catalase concentrations in supernatants were analyzed at 595 nm (Bradford, 1976) by using Shimadzu UV–Visible (1601) spectrophotometer. All experiments were made in triplicate and the results are the means of three different experiments. The amount of catalase immobilized, q (mg g 1), was calculated as follows: q¼
ðC0 −Ce Þ V M
ð2Þ
where C0 and Ce are the initial and equilibrium liquid phase concentrations of catalase (mg L − 1), respectively. V is the volume of the enzyme solution (L) and M is the amount of adsorbent used (g). The kinetics of catalase immobilization onto bentonite and sepiolite was analyzed using pseudo first-order and pseudo second-order kinetic models. Linear form of pseudo-first-order model is expressed as: logðqe −qÞ ¼ logqe −
k1 t 2:303
ð3Þ
where qe and q are the amounts of catalase adsorbed at equilibrium and at time t, respectively (mg g− 1), and k1 is the rate constant (min− 1) (Lagergren, 1898). The linear form of pseudo-second-order equation is expressed as: t 1 1 ¼ þ t qt k2 q2e qe
ð4Þ
116
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
where k2 is the pseudo-second-order rate constant (g mg − 1 min − 1) (Ho and McKay, 1999). 2.9. Surface area measurements BET specific surface areas of bentonite and sepiolite were determined as 77 and 292 m 2 g− 1 respectively by adsorption of N2 at 77 K with Quantachrome Autosorb Automated Gas Sorption System. Characterization data of the samples were given in Table 2. 2.10. SEM analysis Scanning electron microscopy, SEM was carried out in a Philips FEG-XL30 in Darmstadt Technical University, Germany. The surface morphologies of the samples were studied using a scanning electron microscope at an accelerating voltage of 15 kV. The samples that were used were dried and coated with gold before scanning. For SEM, photographs were taken at different magnifications (between 1000× and 5000 ×). 3. Results 3.1. XRD analysis It has been well-known that major clay minerals in bentonites are smectites such as montmorillonite, beidellite, nontronite, hectorite, and saponite which are 2:1 phyllosilicates (Bergaya et al., 2006; Grim, 1968). The clay and non-clay minerals in the bentonite were identified by their characteristic XRD-peaks (Moore and Reynolds, 1997). The XRD patterns of the bentonite (Fig. S1 in supplementary file) showed that it contains a Na–Ca-rich smectite with a d(001) value of 13.06 Å, and also illite, quartz, feldspar, and calcite as impurities. On the other hand, the XRD diffraction pattern of sepiolite was also given in Fig. S2 (in the supplementary file). The XRD pattern of sepiolite contains a sharp reflection at 2θ= 7.34° with a d(001) value of 12.04 Å which is the most characteristic peak of sepiolite (Moore and Reynolds, 1997). On the basis of the XRD peak intensities, other five reflections of sepiolite were also observed with d-values of 4.55, 4.29, 3.71, 3.35 and 3.18 Å. 3.2. Determination of optimum conditions for free and immobilized catalases The effect of temperature on the activities of free and immobilized catalases was studied in 50 mM phosphate buffer (pH = 7). In order to determine the effect of temperature on activities, the reaction of catalase with its substrate, H2O2, was occurred at different temperature values (varying from 15 to 65 °C) while the others remained the same. Relative activities of free and immobilized catalases as a function of temperature were shown in Fig. 1. Maximum activities for free and immobilized catalases were found at 35 °C. According to Fig. 1, relative activity of free catalase decreased rapidly after 35 °C, while the decreasing of the activities of immobilized catalase occurs more slowly. To compare the pH dependencies of the activities of free and immobilized catalases, their relative activities were determined in the pH range 3–12 and the results were shown in Fig. 2. The data showed that optimum pH is shifted from 7 for free catalase to 9 for
Fig. 1. Effect of temperature on activity for free and immobilized catalases. Reactions were performed in phosphate buffer (pH = 7, 50 mM).
immobilized catalase. Besides it is found that the activities of immobilized catalase at alkaline pHs were higher than the activity of free catalase. The effect of ionic strength on the activity of free and immobilized catalases was studied in the ionic strength range 0.01–1 M. In order to do this, the molarity of the buffer solution was changed, while the pH of it was fixed to 7. All these reactions occurred at 35 °C. The results were presented in Fig. 3. The activities of free and immobilized catalases were decreased with increasing ionic strength. Since ionic strength increases the electrostatic interactions, generally enzymes loss their activities at higher ionic conditions. 3.3. Km and Vmax values for free and immobilized catalases In order to determine the relationship between the enzymatic reaction rate and the concentration of the substrate, the reaction between the catalase and H2O2 was observed in optimum conditions (pH = 7, 50 mM, 35 °C) with variable substrate concentrations. The concentration of H2O2 solution varies from 4 mM to 30 mM. To identify the rate of reactions, free or immobilized catalase was added into H2O2 solutions, and the rate of reaction was determined spectrophotometrically. Experimental results were plotted according to the method of Lineweaver–Burk and exhibited in Fig. 4. Km and Vmax values for free and immobilized catalases were given in Table 3. 3.4. Thermal stability of free and immobilized catalases Because of the fact that enzymes used in commercial applications should have thermal stability, thermal stabilities of free and immobilized
Table 2 The properties of bentonite and sepiolite. Sample
XRD analysis d001 (Å)
CEC (meg/100 g clay)
BET specific surface area (m2 g− 1)
Total pore volume (cm3 g− 1)
Average pore diameter (Å)
Bentonite Sepiolite
13.06 12.04
97 82
77 292
0.20 0.57
106 80
Fig. 2. Effect of pH on activity for free and immobilized catalases. Reactions were performed in the buffers (50 mM) at 35 °C.
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
117
Table 3 Adsorption kinetics values for free and immobilized catalases. Adsorption kinetics
Free catalase
Bentonite immobilized catalase
Sepiolite immobilized catalase
Km (mM) Vmax (μmol/min mg)
116.8 30.1
55.6 6.6
49.2 8.7
and 20.1% of their activities, respectively. The enzyme residual activity decreased with the increase of the temperature. Fig. 3. Effect of ionic strength on activity for free and immobilized catalases. Reactions were performed in phosphate buffers of pH = 7 at 35 °C.
catalases were determined at various temperature values for different time intervals. Figs. 5 and 6 indicated the thermal stability of free and immobilized catalases at various temperature values as a function of different preincubation times. As seen in these figures there were no significant activity loss at 15, 25 and 35 °C for free and immobilized catalases. But at 45 °C; although free catalase lost about 50.7% of its initial activity, sepiolite immobilized catalase lost 41.1% and bentonite immobilized catalase lost only 32.6% of their initial activities for 1 h preincubation time. On the other hand, free catalase lost all its initial activity at 65 °C. But sepiolite immobilized catalase and bentonite immobilized catalase remained about 19.1% and 22.3% of their initial activities for 1 h preincubation time, respectively. These results showed that immobilized catalase has a higher thermal stability than free catalase. 3.5. Operational stability of immobilized catalase Immobilized enzymes have a lot of advantages by the side of free ones (Akgöl et al., 2001), for example, they can be easily separated from product solutions and reused (Katchalski-Katzir, 1993). Operational stability of the immobilized catalase was determined at 25 °C for 60 min. It has been found that bentonite immobilized catalase lost about 34.4% of its initial activity after 10 cycles of experiments and sepiolite immobilized catalase lost about 9.8% of its initial activity after 10 cycles of experiments, respectively. 3.6. Storage stability of free and immobilized catalases In order to find the storage stabilities of free and immobilized catalases, the samples were stored in a phosphate buffer (50 mM, pH = 7) at − 15, 4 and 25 °C for a period of 95 days. The activity measurements were carried out in optimum conditions (pH = 7, 50 mM, 35 °C) at specified times. It has been observed that both free and immobilized catalases which were stored at −15 °C were more stable than others stored at 4 °C and 25 °C. At −15 °C, free enzyme lost about 35.8% of its activity after 95 days. In the same interval, bentonite immobilized catalase and sepiolite immobilized catalase lost 28.5%
Fig. 4. Lineweaver–Burk plot for free and immobilized catalases. Reactions were carried out in phosphate buffer (pH = 7, 50 mM) at 35 °C.
3.7. Immobilization kinetics In order to determine the relationship between contact time and catalase immobilization onto bentonite and sepiolite, the equilibrium concentrations were measured at particular times spectrophotometrically and the amounts of catalase adsorbed onto clays were calculated. Equilibrium process was directly correlated with time. Increasing catalase concentration from 50 to 200 μg mL − 1 caused an increase on the adsorption capacities of bentonite and sepiolite from 7.7 mg g − 1 to 33.0 mg g − 1 and 9.6 mg g − 1 to 38.7 mg g − 1, respectively. Pseudo first-order and pseudo second-order kinetic models were used to fit the experimental data of catalase immobilization by clays. The best-fit model was determined based on the linear regression correlation coefficient values. Inasmuch as no linear correlation between the log (qe − q) and t existed, it could be said that pseudo-first-order equation was not convenient for the immobilization of catalase onto clays. The pseudo second-order model was well in line with our experimental results and the related parameters were summarized in Table 4.
3.8. SEM analysis The surface morphologies of clays and catalase loaded clays that were examined by scanning electron microscopy (SEM) were presented in Fig. 7. In the figure, silicate layers with different sizes can be seen and disperse in various directions. It can be added that the distribution of silicate particles is heterogeneous. After loading, in the images of catalase loaded clay, small white grains can be seen. Sepiolite is a chemically hydrated magnesium silicate. Its structure can be described as chain- or needle-like particles instead of plate-like particles of other common clays. The SEM image of sepiolite showed typical fibrous shape type texture. Bundled sepiolite fibers are also noticeable in the image which are marked with an arrow as an example. On the other hand, the morphology of the surface changed greatly after catalase adsorption as in Fig. 7d. This can be due to the fact that catalase particles were accumulated on the surface of the clay sample.
Fig. 5. Stability of free catalase at various temperatures. Enzyme activity was determined in phosphate buffer (pH = 7, 50 mM) at 35 °C.
118
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
Fig. 6. Stability of immobilized catalase at various temperatures. Enzyme activity was determined in phosphate buffer (pH = 7, 50 mM) at 35 °C. a) Bentonite immobilized catalase and b) sepiolite immobilized catalase.
4. Discussion Temperature effects on the activity were studied and the maximum activities were found at 35 °C for free and immobilized catalases. Similar results were also shown by some researchers (Cetinus and Öztop, 2003; Cetinus et al., 2009). According to our results, immobilized catalase showed higher activity than free catalase at high temperatures. It might have been because of the protecting effect of support. It has been also proposed that this result may be caused by changing properties of the enzyme, for example conformational elasticity, by immobilization (Li et al., 2004). Enzymes which lose conformational elasticity by immobilization require a higher temperature to form the suitable conformation that is suitable to bind the substrate molecules. Therefore, immobilized enzymes have a higher optimum temperature than free enzymes in general. Similar results were also presented by Akgöl et al. (2001). Besides, immobilized enzymes had a higher temperature resistance than free enzymes. The increased resistance of the immobilized enzyme in various temperature values indicates good temperature stability of immobilized enzyme. Our results were well in line with the results of reference (Li et al., 2004). The pH of the reaction medium affects the activities of any enzyme. In this study, it was found that the activities of immobilized catalase at alkaline pHs were higher than the activity of free catalase. The covalent bond occurred between support and enzyme may produce the increase of activity at alkaline pHs via providing the stability of enzyme (Sanjay and Sugunan, 2006). The activities of free and immobilized catalases were decreased with increasing ionic
strength as it was expected. Since ionic strength increases the electrostatic interactions, generally enzymes lose their activities at higher ionic conditions. The Lineweaver–Burk plot is used to determine important parameters in enzyme kinetics, such as Km and Vmax. Vmax defines the highest possible velocity when all enzymes are saturated with substrate and Km is a marker for the affinity of enzyme to substrate. Comparison of Km for an enzyme in both the immobilized and free positions provides prediction about the interaction between the enzyme and its support. It was found in the present study that Km values are 116.8 mM for free catalase, 55.6 mM for bentonite immobilized catalase and 49.2 mM for sepiolite immobilized catalase. The Km value of immobilized enzyme which is lower than free enzyme may be caused by the steric regulation of the active site by the support or the regulation of enzyme elasticity essential for substrate binding (Ye et al., 2005). It has been shown that immobilized catalase has a higher thermal stability than free catalase for 1 h preincubation time. This phenomenon had also been observed by other researchers (Cetinus and Öztop, 2003; Costa et al., 2001; Sanjay and Sugunan, 2006; Ye et al., 2005). Besides, the operational and the storage stabilities of immobilized enzymes are very important parameters in industrial usage. Our results showed that bentonite and sepiolite immobilized catalases remained 65.6% and 90.2% of their initial activities after 10 cycles of batch experiments, respectively. Parallel results were also found (Domink et al., 2006). They reported that sol–gel immobilized catalase was found to be stable for 17 batch cycles with no apparent loss in activity. In another study which confirmed this result, it was also reported that the immobilize catalase onto artificial biomembrane retained its original activity even after being employed 160 times in decomposing H2O2 (Tetsuji et al., 2009). The samples which were stored at − 15 °C were more stable than others stored at 4 °C and 25 °C. Although free and immobilized enzymes showed similar trend in terms of activity losses dependent on time, immobilized CAT might be used industrially according to this result. The pseudo second-order model was well in line with our experimental results. It was also found that the sorption capacities at equilibrium were increased with increasing initial catalase concentrations. Consequently, because of lower contact time and remarkable adsorption capacities for catalase, bentonite and sepiolite can be used for adsorption of catalase from aqueous solution. As can also be seen from Fig. 6 the stability of catalase is comparable in the two materials (it is slightly higher in bentonite) although the structure of bentonite and sepiolite is distinctively different. Sepiolite has silanol groups at the external surface of the silicate, which are usually accessible to organic species, acting as neutral adsorption sites. Sorption takes place on neutral sites and neutral complexes that are formed through sorption of an organic cation on a negative site. On the other hand, bentonite used in this study is a Na-Ca-rich smectite, the isomorphous substitution of Al 3 + for Si4 + in the tetrahedral layer and Mg2 + for Al3 + in the octahedral layer, which results in a net negative surface charge on the bentonite. This charge imbalance is offset by exchangeable cations (Na +, Ca 2 +, etc.) at the bentonite surface. The layered structure of the clay expands after wetting. Na+ and Ca2 + are strongly hydrated in the presence of water, resulting in a hydrophilic
Table 4 The pseudo-second-order parameters for adsorption of catalase onto bentonite and sepiolite at different initial catalase concentrations. Bentonite Catalase concentration (μg mL R2 k2 (g mg− 1 min− 1) q2(exp) (mg g− 1) q2(cal) (mg g− 1)
−1
)
50 0.9953 0.049 8.83 9.07
Sepiolite 100 0.9993 0.063 18.35 18.42
200 0.9993 0.023 35.52 35.84
50 0.9913 0.068 9.61 9.53
100 0.9999 0.076 19.39 19.49
200 0.9999 0.065 38.65 38.76
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
119
Fig. 7. SEM images of a) bentonite, b) catalase immobilized bentonite, c) sepiolite, and d) catalase immobilized sepiolite.
environment at the bentonite surface. Isomorphic substitution, cation exchange mechanism (i.e. CEC is relatively higher than sepiolite) and also layer charge which is one of the most important characteristics of 2:1 phyllosilicates may be responsible for the additional effectiveness of stability as compared with sepiolite. 5. Conclusions The present paper demonstrates the immobilization of catalase onto clays, the characterization of immobilization process and the activity measurements of free and immobilized catalases. The experimental results showed that temperature, pH and the ionic strength of the medium could have an important effect on the activity of free and immobilized catalases. It was observed that thermal and storage stabilities of catalase increased by immobilization. And also immobilized catalase had an operational stability. The better resistance of immobilized catalase against the environmental changes indicates that immobilization is a good way to use enzymes for industrial applications. Since these natural clays have some required characteristics that are important in immobilization, they can be recommended as a carrier for enzyme immobilization. Acknowledgments The author (S. Cengiz) was financially supported by TUBITAK (the Scientific and Technological Research Council of Turkey) scholarship. Authors would like to thank Prof. Dr. Peter Claus, Darmstadt Technical University/Germany for SEM and BET analyses. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.clay.2012.06.004. References Aebi, H.E., 1974. Catalase, In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, 3rd ed. Verlag Chemie, Weinheim, pp. 672–684. Akgöl, S., Kaçar, Y., Özkara, S., Yavuz, H., Denizli, A., Arica, M.Y., 2001. Immobilization of catalase via adsorption onto L-histidine grafted functional pHEMA based membrane. Journal of Molecular Catalysis B: Enzymatic 15, 197–206.
Akgöl, S., Yavuz, H., Şenel, S., Denizli, A., 2003. Glucose oxidase and catalase adsorption onto Cibacron Blue F3GA-attached microporous polyamide hollow-fibres. Reactive and Functional Polymers 55, 45–51. Alkan, S., Ceylan, H., Arslan, O., 2005. Bentonite-supported catalase. Journal of the Serbian Chemical Society 70 (5), 721–726. Atkins, P., Paula, J., 2006. Physical Chemistry. Oxford Univ. Press, New York. 840 pp. Bergaya, F., Theng, B.K.G., Lagaly, G., 2006. Handbook of Clay Science, Development in Clay Science, vol. 1. Elsevier, Amsterdam. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Cabana, H., Alexandre, C., Agathos, S.N., Jones, J.P., 2009. Immobilization of lactase from the white rot fungus Coriolopsis polyzona and use of the immobilized biocatalyst for the continuous elimination of endocrine disrupting chemicals. Bioresource Technology 100, 3447–3458. Caillou, S., Gerin, P.A., Nonckreman, C.J., Fleith, S., Dupont-Gillain, C.C., Landoulsi, J., Pancera, S.M., Genet, M.J., Rouxhet, P.G., 2008. Enzymes at solid surfaces: nature of the interfaces and physico-chemical processes. Electrochimica Acta 54, 116–122. Carrasco, M.S., Rad, S.C., Gonzalez-Carcedo, 1995. Immobilization of alkaline phosphatase by sorption on Na-sepiolite. Bioresource Technology 51, 175–181. Cetinus, S.A., Öztop, H.N., 2003. Immobilization of catalase into chemically crosslinked chitosan beads. Enzyme and Microbial Technology 32, 889–894. Cetinus, S.A., Şahin, E., Saraydin, D., 2009. Preparation of Cu(II) adsorbed chitosan beads for catalase immobilization. Food Chemistry 114, 962–969. Chang, M.Y., Juang, R.S., 2007. Use of chitosan–clay composite as immobilization support for improved activity and stability of β-glucosidase. Biochemical Engineering Journal 35, 93–98. Choi, M.M.F., Yiu, T.P., 2004. Immobilization of beef liver catalase on eggshell membrane for fabrication of hydrogen peroxide biosensor. Enzyme and Microbial Technology 34, 41–47. Costa, S.A., Tzanov, T., Paar, A., Gudelj, M., Gübitz, G.M., Cavaco-Paulo, A., 2001. Immobilization of catalases from Bacillus SF on alumina for the treatment of textile bleaching effluents. Enzyme and Microbial Technology 28, 815–819. Costa, S.A., Tzanov, T., Carneiro, A.F., Paar, A., Gübitz, G.M., Cavaco-Paulo, A., 2002. Studies of stabilization of native catalase using additives. Enzyme and Microbial Technology 30, 387–391. Demirbas, O., Alkan, M., 2011. Thermodynamics, kinetics and adsorption properties of some biomolecules onto mineral surfaces. In: Piraján, Juan Carlos Moreno (Ed.), Thermodynamics — Kinetics of Dynamic Systems. InTech, pp. 315–330. Domink, L., Lohmann, J., Legge, R.L., 2006. Immobilization of bovine catalase in sol–gels. Enzyme and Microbial Technology 39, 626–633. Eberhardt, A.M., Pedroni, V., Volpe, M., Ferreira, M.L., 2004. Immobilization of catalase from Aspergillus niger on inorganic and biopolymeric supports for H2O2 decomposition. Applied Catalysis B: Environmental 47, 153–163. Grim, R.E., 1968. Clay Mineralogy, 2nd ed. McGraw-Hill, New York. Ho, Y.S., McKay, G., 1999. Pseudo second-order model for sorption processes. Process Biochemistry 34, 451–465. Itoh, T., Ishii, R., Matsuura, S., Hamakawa, S., Hanaoka, T., Tsunoda, T., Mizuguchi, J., Mizukami, F., 2009a. Catalase encapsulated in mesoporous silica and its performance. Biochemical Engineering Journal 44, 167–173. Itoh, T., Ishii, R., Hanaoka, T., Hasegawa, Y., Mizuguchi, J., Shiomib, T., Shimomurab, T., Yamaguchi, A., Kaneda, H., Teramae, N., Mizukami, F., 2009b. Encapsulation of
120
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
catalase into nanochannels of an inorganic composite membrane. Journal of Molecular Catalysis B: Enzymatic 57, 183–187. Katchalski-Katzir, E., 1993. Immobilized enzymes — learning from past successes and failures. Trends in Biotechnology 11, 471–478. Kilara, A., 1981. Immobilized proteases and lipases. Process 25–27. Krajewska, B., 2009. Ureases. II. Properties and their customizing by enzyme immobilizations: a review. Journal of Molecular Catalysis B: Enzymatic 59, 22–40. Kubal, B.S., D'Souza, S.F., 2004. Immobilization of catalase by entrapment of permeabilized yeast cells in hen egg white using glutaraldehyde. Journal of Biochemical and Biophysical Methods 59, 61–64. Kumar, G.S.V., Kumar, K.S., 2005. Immobilization of catalase on a novel polymer support, crosslinked polystyrene ethylene glycol acrylate resin: role of the macromolecular matrix on enzyme activity. Journal of Applied Polymer Science 97, 8–19. Lagergren, S., 1898. Zur theorie der sogenannten adsorption gelöster stoffe. Kungliga Svenska Vetenskapsakademiens Handlingar 24 (4), 1–39. Li, S., Hu, J., Liu, B., 2004. Use of chemically modified PMMA microspheres for enzyme immobilization. BioSystems 77, 25–32. Moore, D.M., Reynolds Jr., R.C., 1997. X-ray Diffraction and the Identification and Analysis of Minerals. Oxford University Press, New York. Öztürk, N., Tabak, A., Akgöl, S., Denizli, A., 2008. Reversible immobilization of catalase by using a novel bentonite–cysteine (Bent–Cys) microcomposite affinity sorbents. Colloids and Surfaces A: Physicochemical and Engineering Aspects 322, 148–154. Popa, M., Bajan, N., Popa, A.A., Verestiuc, L., 2006. The preparation, characterization and properties of catalase immobilized on crosslinked gellan. Journal of Macromolecular Science, Part A Pure and Applied Chemistry 43, 355–367. Sanjay, G., Sugunan, S., 2006. Enhanced pH and thermal stabilities of invertase immobilized on montmorillonite K-10. Food Chemistry 94, 573–579.
Sedaghat, M.E., Ghiaci, M., Aghaei, H., Soleimanian-Zad, S., 2009. Enzyme immobilization. Part 4. Immobilization of alkaline phosphatase on Na-sepiolite and modified sepiolite. Applied Clay Science 46, 131–135. Shentu, J., Wu, J., Song, W., Jia, Z., 2005. Chitosan microspheres as immobilized dye affinity support for catalase adsorption. International Journal of Biological Macromolecules 37, 42–46. Tarhan, L., 1995. Use of immobilised catalase to remove H2O2 used in the sterilisation of milk. Process Biochemistry 30 (7), 623–628. Tarhan, L., Telefoncu, A., 1992. Effect of enzyme ratio on the properties of glucose oxidase and catalase immobilized on modified perlite. Process Biochemistry 27 (1), 11–15. Tetsuji, I., Ryo, I., Takaaki, H., Yasuhisa, H., Junko, M., Toru, S., Takeshi, S., Akira, Y., Hideaki, K., Norio, T., Fujio, M., 2009. Encapsulation of catalase into nanochannels of an inorganic composite membrane. Journal of Molecular Catalysis B: Enzymatic 57, 183–187. Tukel, S.S., Alptekin, O., 2004. Immobilization and kinetics of catalase onto magnesium silicate. Process Biochemistry 39, 2149–2155. Wang, Q., Xuan Li, C., Fan, X., Wang, P., Cui, L., 2008. Immobilization of catalase on cotton fabric oxidized by sodium periodate. Biocatalysis and Biotransformation 26 (5), 437–443. Ye, P., Xu, Z.K., Che, A.F., Wu, J., Seta, P., 2005. Chitosan-tethered poly (acrylonitrileco-maleic acid) hollow fiber membrane for lipase immobilization. Biomaterials 26, 6394–6403.
Contents lists available at SciVerse ScienceDirect
Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Research paper
Bentonite and sepiolite as supporting media: Immobilization of catalase Sevilay Cengiz a, Levent Çavaş b, Kadir Yurdakoç b,⁎ a b
Akdeniz University, Faculty of Arts and Sciences, Department of Chemistry, 07058 Antalya, Turkey Dokuz Eylul University, Faculty of Science, Department of Chemistry, Tinaztepe Campus, 35160 Buca/Izmir, Turkey
a r t i c l e
i n f o
Article history: Received 9 June 2011 Received in revised form 4 June 2012 Accepted 5 June 2012 Available online 21 July 2012 Keywords: Bentonite Sepiolite Clay Adsorption Immobilization Catalase
a b s t r a c t Immobilization of catalase onto bentonite and sepiolite was studied as a function of pH, temperature and ionic strength for free and immobilized enzyme activities. The optimum temperature, pH and ionic strength for free and immobilized catalases were 35 °C, pH=7 and 50 mM (for free catalase) and 35 °C, pH=9 and 50 mM (for both immobilized catalases), respectively. Km and Vmax values were 116.8 mM and 30.1 μmol min−1 mg protein− 1 for free catalase, 55.6 mM, Vmax =6.6 μmol min−1 mg protein− 1 for bentonite supported catalase and Km = 49.2 mM, Vmax = 8.7 μmol min− 1 mg protein− 1 for sepiolite supported catalase. The storage stabilities of immobilized catalase were higher than free catalase. Free catalase lost 50.7% of its initial activity, while sepiolite immobilized catalase lost 41.1% and bentonite immobilized catalase lost only 32.6% of their initial activities for 1 h preincubation time at 45 °C. Both immobilized catalases showed an operational stability and for a period of 10 cycles of experiments they lost only 34.4% and 9.8% of their initial activities for bentonite and sepiolite immobilized catalases, respectively. Batch adsorption studies were also carried out to determine the kinetic parameters. The pseudo second order model provided the best correlation with the experimental results. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Enzymes have useful applications in various industrial fields such as biotechnological, pharmaceutical and food industries (Li et al., 2004). Enzymes have taken lots of interests from a lot of researchers owing to unique properties such as catalytic efficiency, specificity and mild conditions of operation. Although these properties enable enzymes to catalyze a series of biotransformation reactions, their usage is restricted with some problems including reusing of free enzymes, limited stability against high temperatures, pHs, ionic strengths and inhibitory sensitivities (Krajewska, 2009). There are some methods to cope with these problems. One of them is immobilization that can be made by some techniques such as adsorption, covalent binding, and entrapment vb. Among these, adsorption of enzymes onto solid supports is preferred in order to enhance the industrial utility, to improve their stability and to provide their continuous use (Cabana et al., 2009). The basic requirements of support materials which are used for enzyme immobilization are mechanical and thermal strength, microbial resistance, and non-toxicity, and regenerability, low cost and also large surface area for enzymic reactions (Kilara, 1981; Li et al., 2004). Up to now, various types of support materials such as chitosan (Shentu et al., 2005), a crosslinked macromolecular carrier of a polysaccharide structure (gellan) (Popa et al., 2006), chitosan–clay composite (Chang and Juang, 2007), Cu(II) adsorbed
⁎ Corresponding author. Tel.: + 90 232 3018695; fax: + 90 232 4534188. E-mail address: [email protected] (K. Yurdakoç). 0169-1317/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2012.06.004
chitosan beads (Cetinus et al., 2009), microporous polyamide hollowfibers (Akgöl et al., 2003), a novel bentonite–cysteine (Bent–Cys) microcomposite (Öztürk et al., 2008), magnesium silicate (Tukel and Alptekin, 2004), eggshell membrane (Choi and Yiu, 2004), alumina and cellulose (Eberhardt et al., 2004), crosslinked polystyrene ethylene glycol acrylate resin (CLPSER) (Kumar and Kumar, 2005), tetraethoxyorthosilicate (TEOS) based sol–gels (Domink et al., 2006), surface-modified carbon blacks (Caillou et al., 2008), cotton fabric (Wang et al., 2008), mesoporous silica (Itoh et al., 2009a), silica–alumina inorganic composite membrane (Itoh et al., 2009b), bentonite (Alkan et al., 2005), and modified perlite (Tarhan and Telefoncu, 1992) have been used for catalase immobilization. Immobilization of alkaline phosphatase on Na-sepiolite and modified sepiolite was studied in details (Carrasco et al., 1995; Sedaghat et al., 2009). Adsorption and electrokinetic properties of casein and catalase onto sepiolite were also investigated (Demirbas and Alkan, 2011). However, adsorption of catalase onto sepiolite, or usage of sepiolite for immobilization of catalase was still not studied so much, because there is very limited information in the literature. In the light of the knowledge mentioned above bentonite and especially sepiolite were chosen in this study as a support because of their low cost, high specific surface area, chemical, mechanical and thermal stability, porous structure and high adsorption capacities. Catalase (EC 1.11.1.6) that consists of four protein subunits is an abundant native enzyme, which decomposes H2O2 to water and oxygen (Aebi, 1974). Immobilized catalase is used in many industrial processes such as textile (Costa et al., 2002; Eberhardt et al., 2004)
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
and food industries (Akgöl et al., 2001; Kubal and D'Souza, 2004), treatment of waste waters and also in contact-lenses solutions, cosmetics and pharmaceutical formulations (Choi and Yiu, 2004) to determine the quantity of H2O2 or to remove the excess of H2O2 (Tarhan, 1995). Therefore, the present study aimed to investigate the immobilization characteristics of catalase onto bentonite and sepiolite from Turkey. Besides the kinetic parameters, thermal, storage and operational stabilities of free and immobilized catalases will also be determined and compared to each other.
115
by catalase at 240 nm. The amount of H2O2 converted into H2O and O2 in 1 min under standard conditions is accepted as the enzyme reaction rate. The optimum temperature, pH and ionic strength values were determined for free and immobilized enzymes. Michaelis constants (Km) and maximum rate (Vmax) values for free and immobilized catalases were also determined from the slope of the x-axis and the intercept of the y-axis, respectively according to below equation (Atkins and Paula, 2006). 1 1 KM 1 ¼ þ : ⋅ V V max V max ½S0
ð1Þ
2. Materials and methods 2.5. Determination of thermal stability of catalase
2.1. Clays and catalase Bentonite, a sodium–calcium-rich smectite (NaCaS) of light yellowish green color and sepiolite (S) were obtained from Enez/Edirne and Eskisehir-Turkey, respectively and used as supporting agents in the experiments. The properties of the clays were summarized in Table 1. Catalase was obtained from Sigma (Sigma Cat. No. C 9322) and used without further purification and treatment. In the preparation of catalase solution, phosphate buffer (pH = 7, 50 mM) was used. 2.2. XRD analysis X-ray diffraction patterns of the clays (XRD) were obtained with oriented mounts, in a Phillips X'Pert Pro instrument using Cu Kα radiation with a wavelength of 1.5418 Å, and range for bentonite 2θ = 3–10° and 3–70°, for sepiolite 2θ = 5–30°. 2.3. Immobilization of catalase onto clays 5 mg of bentonite or sepiolite was added into 1 mL catalase solutions that have varying concentrations between 30 and 200 μg mL− 1. The samples were agitated at 105 rpm until these reach the equilibrium conditions in a temperature controlled shaker (Memmert). The immobilized catalase was separated from the residual aliquot by centrifugation at 5000 rpm for 5 min at +4 °C (Hettich, R21). The amount of catalase immobilized onto clays was calculated by measuring the initial and equilibrium concentrations of catalase in the medium by the method of Bradford (1976). The pH of the immobilization medium was varied from 3 to 12 by using different buffer systems (50 mM CH3COOH for pH =3–5; 50 mM KH2PO4–K2HPO4 for pH = 7–9 and 50 mM NaOH– KCl for pH = 12). The temperature and the ionic strength of the immobilization medium were varied from 15 °C to 65 °C and from 0.01 to 1 M, respectively. 2.4. Catalase activity assay The activity of free and immobilized catalases was determined spectrophotometrically (Shimadzu UV–Visible 1601) at 240 nm with Aebi method (Aebi, 1974). This method is based on the principle that the absorbance will decrease due to the decomposition of hydrogen peroxide
Table 1 Chemical composition of bentonite and sepiolite (%). Component
Bentonite
Sepiolite
SiO2 Al2O3 Fe2O3 MgO CaO Na2O K2O Sulfate Ignition loss
57.8 19.2 3.6 2.1 4.2 2.7 1.4 – 7.5
48.0 0.5 0.4 24.0 4.0 0.02 0.03 0.02 23.0
The effect of temperature on the activities of free and immobilized catalases was determined at 35 °C, thereafter incubating the samples in phosphate buffers (pH = 7) at various temperatures (15–65 °C) for specified times. 2.6. Determination of storage stability of catalase In order to find out the storage stabilities of free and immobilized catalases at various temperatures (−18 °C, 4 °C and 25 °C), the samples were incubated in phosphate buffer (pH = 7) for days and their activities were found periodically at 35 °C. 2.7. Determination of operational stability of catalase To identify the operational stabilities of immobilized catalase, prepared samples were added into substrate solution and stirred slowly. Then the solution was separated from immobilized enzyme and its absorbance was measured at 240 nm. Immobilized catalase was washed with phosphate buffer (pH = 7), then the same experiments were repeated several times. 2.8. Immobilization kinetics The immobilization experiments were done with 5 mg of the clays at 35 °C. The catalase concentrations in supernatants were analyzed at 595 nm (Bradford, 1976) by using Shimadzu UV–Visible (1601) spectrophotometer. All experiments were made in triplicate and the results are the means of three different experiments. The amount of catalase immobilized, q (mg g 1), was calculated as follows: q¼
ðC0 −Ce Þ V M
ð2Þ
where C0 and Ce are the initial and equilibrium liquid phase concentrations of catalase (mg L − 1), respectively. V is the volume of the enzyme solution (L) and M is the amount of adsorbent used (g). The kinetics of catalase immobilization onto bentonite and sepiolite was analyzed using pseudo first-order and pseudo second-order kinetic models. Linear form of pseudo-first-order model is expressed as: logðqe −qÞ ¼ logqe −
k1 t 2:303
ð3Þ
where qe and q are the amounts of catalase adsorbed at equilibrium and at time t, respectively (mg g− 1), and k1 is the rate constant (min− 1) (Lagergren, 1898). The linear form of pseudo-second-order equation is expressed as: t 1 1 ¼ þ t qt k2 q2e qe
ð4Þ
116
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
where k2 is the pseudo-second-order rate constant (g mg − 1 min − 1) (Ho and McKay, 1999). 2.9. Surface area measurements BET specific surface areas of bentonite and sepiolite were determined as 77 and 292 m 2 g− 1 respectively by adsorption of N2 at 77 K with Quantachrome Autosorb Automated Gas Sorption System. Characterization data of the samples were given in Table 2. 2.10. SEM analysis Scanning electron microscopy, SEM was carried out in a Philips FEG-XL30 in Darmstadt Technical University, Germany. The surface morphologies of the samples were studied using a scanning electron microscope at an accelerating voltage of 15 kV. The samples that were used were dried and coated with gold before scanning. For SEM, photographs were taken at different magnifications (between 1000× and 5000 ×). 3. Results 3.1. XRD analysis It has been well-known that major clay minerals in bentonites are smectites such as montmorillonite, beidellite, nontronite, hectorite, and saponite which are 2:1 phyllosilicates (Bergaya et al., 2006; Grim, 1968). The clay and non-clay minerals in the bentonite were identified by their characteristic XRD-peaks (Moore and Reynolds, 1997). The XRD patterns of the bentonite (Fig. S1 in supplementary file) showed that it contains a Na–Ca-rich smectite with a d(001) value of 13.06 Å, and also illite, quartz, feldspar, and calcite as impurities. On the other hand, the XRD diffraction pattern of sepiolite was also given in Fig. S2 (in the supplementary file). The XRD pattern of sepiolite contains a sharp reflection at 2θ= 7.34° with a d(001) value of 12.04 Å which is the most characteristic peak of sepiolite (Moore and Reynolds, 1997). On the basis of the XRD peak intensities, other five reflections of sepiolite were also observed with d-values of 4.55, 4.29, 3.71, 3.35 and 3.18 Å. 3.2. Determination of optimum conditions for free and immobilized catalases The effect of temperature on the activities of free and immobilized catalases was studied in 50 mM phosphate buffer (pH = 7). In order to determine the effect of temperature on activities, the reaction of catalase with its substrate, H2O2, was occurred at different temperature values (varying from 15 to 65 °C) while the others remained the same. Relative activities of free and immobilized catalases as a function of temperature were shown in Fig. 1. Maximum activities for free and immobilized catalases were found at 35 °C. According to Fig. 1, relative activity of free catalase decreased rapidly after 35 °C, while the decreasing of the activities of immobilized catalase occurs more slowly. To compare the pH dependencies of the activities of free and immobilized catalases, their relative activities were determined in the pH range 3–12 and the results were shown in Fig. 2. The data showed that optimum pH is shifted from 7 for free catalase to 9 for
Fig. 1. Effect of temperature on activity for free and immobilized catalases. Reactions were performed in phosphate buffer (pH = 7, 50 mM).
immobilized catalase. Besides it is found that the activities of immobilized catalase at alkaline pHs were higher than the activity of free catalase. The effect of ionic strength on the activity of free and immobilized catalases was studied in the ionic strength range 0.01–1 M. In order to do this, the molarity of the buffer solution was changed, while the pH of it was fixed to 7. All these reactions occurred at 35 °C. The results were presented in Fig. 3. The activities of free and immobilized catalases were decreased with increasing ionic strength. Since ionic strength increases the electrostatic interactions, generally enzymes loss their activities at higher ionic conditions. 3.3. Km and Vmax values for free and immobilized catalases In order to determine the relationship between the enzymatic reaction rate and the concentration of the substrate, the reaction between the catalase and H2O2 was observed in optimum conditions (pH = 7, 50 mM, 35 °C) with variable substrate concentrations. The concentration of H2O2 solution varies from 4 mM to 30 mM. To identify the rate of reactions, free or immobilized catalase was added into H2O2 solutions, and the rate of reaction was determined spectrophotometrically. Experimental results were plotted according to the method of Lineweaver–Burk and exhibited in Fig. 4. Km and Vmax values for free and immobilized catalases were given in Table 3. 3.4. Thermal stability of free and immobilized catalases Because of the fact that enzymes used in commercial applications should have thermal stability, thermal stabilities of free and immobilized
Table 2 The properties of bentonite and sepiolite. Sample
XRD analysis d001 (Å)
CEC (meg/100 g clay)
BET specific surface area (m2 g− 1)
Total pore volume (cm3 g− 1)
Average pore diameter (Å)
Bentonite Sepiolite
13.06 12.04
97 82
77 292
0.20 0.57
106 80
Fig. 2. Effect of pH on activity for free and immobilized catalases. Reactions were performed in the buffers (50 mM) at 35 °C.
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
117
Table 3 Adsorption kinetics values for free and immobilized catalases. Adsorption kinetics
Free catalase
Bentonite immobilized catalase
Sepiolite immobilized catalase
Km (mM) Vmax (μmol/min mg)
116.8 30.1
55.6 6.6
49.2 8.7
and 20.1% of their activities, respectively. The enzyme residual activity decreased with the increase of the temperature. Fig. 3. Effect of ionic strength on activity for free and immobilized catalases. Reactions were performed in phosphate buffers of pH = 7 at 35 °C.
catalases were determined at various temperature values for different time intervals. Figs. 5 and 6 indicated the thermal stability of free and immobilized catalases at various temperature values as a function of different preincubation times. As seen in these figures there were no significant activity loss at 15, 25 and 35 °C for free and immobilized catalases. But at 45 °C; although free catalase lost about 50.7% of its initial activity, sepiolite immobilized catalase lost 41.1% and bentonite immobilized catalase lost only 32.6% of their initial activities for 1 h preincubation time. On the other hand, free catalase lost all its initial activity at 65 °C. But sepiolite immobilized catalase and bentonite immobilized catalase remained about 19.1% and 22.3% of their initial activities for 1 h preincubation time, respectively. These results showed that immobilized catalase has a higher thermal stability than free catalase. 3.5. Operational stability of immobilized catalase Immobilized enzymes have a lot of advantages by the side of free ones (Akgöl et al., 2001), for example, they can be easily separated from product solutions and reused (Katchalski-Katzir, 1993). Operational stability of the immobilized catalase was determined at 25 °C for 60 min. It has been found that bentonite immobilized catalase lost about 34.4% of its initial activity after 10 cycles of experiments and sepiolite immobilized catalase lost about 9.8% of its initial activity after 10 cycles of experiments, respectively. 3.6. Storage stability of free and immobilized catalases In order to find the storage stabilities of free and immobilized catalases, the samples were stored in a phosphate buffer (50 mM, pH = 7) at − 15, 4 and 25 °C for a period of 95 days. The activity measurements were carried out in optimum conditions (pH = 7, 50 mM, 35 °C) at specified times. It has been observed that both free and immobilized catalases which were stored at −15 °C were more stable than others stored at 4 °C and 25 °C. At −15 °C, free enzyme lost about 35.8% of its activity after 95 days. In the same interval, bentonite immobilized catalase and sepiolite immobilized catalase lost 28.5%
Fig. 4. Lineweaver–Burk plot for free and immobilized catalases. Reactions were carried out in phosphate buffer (pH = 7, 50 mM) at 35 °C.
3.7. Immobilization kinetics In order to determine the relationship between contact time and catalase immobilization onto bentonite and sepiolite, the equilibrium concentrations were measured at particular times spectrophotometrically and the amounts of catalase adsorbed onto clays were calculated. Equilibrium process was directly correlated with time. Increasing catalase concentration from 50 to 200 μg mL − 1 caused an increase on the adsorption capacities of bentonite and sepiolite from 7.7 mg g − 1 to 33.0 mg g − 1 and 9.6 mg g − 1 to 38.7 mg g − 1, respectively. Pseudo first-order and pseudo second-order kinetic models were used to fit the experimental data of catalase immobilization by clays. The best-fit model was determined based on the linear regression correlation coefficient values. Inasmuch as no linear correlation between the log (qe − q) and t existed, it could be said that pseudo-first-order equation was not convenient for the immobilization of catalase onto clays. The pseudo second-order model was well in line with our experimental results and the related parameters were summarized in Table 4.
3.8. SEM analysis The surface morphologies of clays and catalase loaded clays that were examined by scanning electron microscopy (SEM) were presented in Fig. 7. In the figure, silicate layers with different sizes can be seen and disperse in various directions. It can be added that the distribution of silicate particles is heterogeneous. After loading, in the images of catalase loaded clay, small white grains can be seen. Sepiolite is a chemically hydrated magnesium silicate. Its structure can be described as chain- or needle-like particles instead of plate-like particles of other common clays. The SEM image of sepiolite showed typical fibrous shape type texture. Bundled sepiolite fibers are also noticeable in the image which are marked with an arrow as an example. On the other hand, the morphology of the surface changed greatly after catalase adsorption as in Fig. 7d. This can be due to the fact that catalase particles were accumulated on the surface of the clay sample.
Fig. 5. Stability of free catalase at various temperatures. Enzyme activity was determined in phosphate buffer (pH = 7, 50 mM) at 35 °C.
118
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
Fig. 6. Stability of immobilized catalase at various temperatures. Enzyme activity was determined in phosphate buffer (pH = 7, 50 mM) at 35 °C. a) Bentonite immobilized catalase and b) sepiolite immobilized catalase.
4. Discussion Temperature effects on the activity were studied and the maximum activities were found at 35 °C for free and immobilized catalases. Similar results were also shown by some researchers (Cetinus and Öztop, 2003; Cetinus et al., 2009). According to our results, immobilized catalase showed higher activity than free catalase at high temperatures. It might have been because of the protecting effect of support. It has been also proposed that this result may be caused by changing properties of the enzyme, for example conformational elasticity, by immobilization (Li et al., 2004). Enzymes which lose conformational elasticity by immobilization require a higher temperature to form the suitable conformation that is suitable to bind the substrate molecules. Therefore, immobilized enzymes have a higher optimum temperature than free enzymes in general. Similar results were also presented by Akgöl et al. (2001). Besides, immobilized enzymes had a higher temperature resistance than free enzymes. The increased resistance of the immobilized enzyme in various temperature values indicates good temperature stability of immobilized enzyme. Our results were well in line with the results of reference (Li et al., 2004). The pH of the reaction medium affects the activities of any enzyme. In this study, it was found that the activities of immobilized catalase at alkaline pHs were higher than the activity of free catalase. The covalent bond occurred between support and enzyme may produce the increase of activity at alkaline pHs via providing the stability of enzyme (Sanjay and Sugunan, 2006). The activities of free and immobilized catalases were decreased with increasing ionic
strength as it was expected. Since ionic strength increases the electrostatic interactions, generally enzymes lose their activities at higher ionic conditions. The Lineweaver–Burk plot is used to determine important parameters in enzyme kinetics, such as Km and Vmax. Vmax defines the highest possible velocity when all enzymes are saturated with substrate and Km is a marker for the affinity of enzyme to substrate. Comparison of Km for an enzyme in both the immobilized and free positions provides prediction about the interaction between the enzyme and its support. It was found in the present study that Km values are 116.8 mM for free catalase, 55.6 mM for bentonite immobilized catalase and 49.2 mM for sepiolite immobilized catalase. The Km value of immobilized enzyme which is lower than free enzyme may be caused by the steric regulation of the active site by the support or the regulation of enzyme elasticity essential for substrate binding (Ye et al., 2005). It has been shown that immobilized catalase has a higher thermal stability than free catalase for 1 h preincubation time. This phenomenon had also been observed by other researchers (Cetinus and Öztop, 2003; Costa et al., 2001; Sanjay and Sugunan, 2006; Ye et al., 2005). Besides, the operational and the storage stabilities of immobilized enzymes are very important parameters in industrial usage. Our results showed that bentonite and sepiolite immobilized catalases remained 65.6% and 90.2% of their initial activities after 10 cycles of batch experiments, respectively. Parallel results were also found (Domink et al., 2006). They reported that sol–gel immobilized catalase was found to be stable for 17 batch cycles with no apparent loss in activity. In another study which confirmed this result, it was also reported that the immobilize catalase onto artificial biomembrane retained its original activity even after being employed 160 times in decomposing H2O2 (Tetsuji et al., 2009). The samples which were stored at − 15 °C were more stable than others stored at 4 °C and 25 °C. Although free and immobilized enzymes showed similar trend in terms of activity losses dependent on time, immobilized CAT might be used industrially according to this result. The pseudo second-order model was well in line with our experimental results. It was also found that the sorption capacities at equilibrium were increased with increasing initial catalase concentrations. Consequently, because of lower contact time and remarkable adsorption capacities for catalase, bentonite and sepiolite can be used for adsorption of catalase from aqueous solution. As can also be seen from Fig. 6 the stability of catalase is comparable in the two materials (it is slightly higher in bentonite) although the structure of bentonite and sepiolite is distinctively different. Sepiolite has silanol groups at the external surface of the silicate, which are usually accessible to organic species, acting as neutral adsorption sites. Sorption takes place on neutral sites and neutral complexes that are formed through sorption of an organic cation on a negative site. On the other hand, bentonite used in this study is a Na-Ca-rich smectite, the isomorphous substitution of Al 3 + for Si4 + in the tetrahedral layer and Mg2 + for Al3 + in the octahedral layer, which results in a net negative surface charge on the bentonite. This charge imbalance is offset by exchangeable cations (Na +, Ca 2 +, etc.) at the bentonite surface. The layered structure of the clay expands after wetting. Na+ and Ca2 + are strongly hydrated in the presence of water, resulting in a hydrophilic
Table 4 The pseudo-second-order parameters for adsorption of catalase onto bentonite and sepiolite at different initial catalase concentrations. Bentonite Catalase concentration (μg mL R2 k2 (g mg− 1 min− 1) q2(exp) (mg g− 1) q2(cal) (mg g− 1)
−1
)
50 0.9953 0.049 8.83 9.07
Sepiolite 100 0.9993 0.063 18.35 18.42
200 0.9993 0.023 35.52 35.84
50 0.9913 0.068 9.61 9.53
100 0.9999 0.076 19.39 19.49
200 0.9999 0.065 38.65 38.76
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
119
Fig. 7. SEM images of a) bentonite, b) catalase immobilized bentonite, c) sepiolite, and d) catalase immobilized sepiolite.
environment at the bentonite surface. Isomorphic substitution, cation exchange mechanism (i.e. CEC is relatively higher than sepiolite) and also layer charge which is one of the most important characteristics of 2:1 phyllosilicates may be responsible for the additional effectiveness of stability as compared with sepiolite. 5. Conclusions The present paper demonstrates the immobilization of catalase onto clays, the characterization of immobilization process and the activity measurements of free and immobilized catalases. The experimental results showed that temperature, pH and the ionic strength of the medium could have an important effect on the activity of free and immobilized catalases. It was observed that thermal and storage stabilities of catalase increased by immobilization. And also immobilized catalase had an operational stability. The better resistance of immobilized catalase against the environmental changes indicates that immobilization is a good way to use enzymes for industrial applications. Since these natural clays have some required characteristics that are important in immobilization, they can be recommended as a carrier for enzyme immobilization. Acknowledgments The author (S. Cengiz) was financially supported by TUBITAK (the Scientific and Technological Research Council of Turkey) scholarship. Authors would like to thank Prof. Dr. Peter Claus, Darmstadt Technical University/Germany for SEM and BET analyses. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.clay.2012.06.004. References Aebi, H.E., 1974. Catalase, In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, 3rd ed. Verlag Chemie, Weinheim, pp. 672–684. Akgöl, S., Kaçar, Y., Özkara, S., Yavuz, H., Denizli, A., Arica, M.Y., 2001. Immobilization of catalase via adsorption onto L-histidine grafted functional pHEMA based membrane. Journal of Molecular Catalysis B: Enzymatic 15, 197–206.
Akgöl, S., Yavuz, H., Şenel, S., Denizli, A., 2003. Glucose oxidase and catalase adsorption onto Cibacron Blue F3GA-attached microporous polyamide hollow-fibres. Reactive and Functional Polymers 55, 45–51. Alkan, S., Ceylan, H., Arslan, O., 2005. Bentonite-supported catalase. Journal of the Serbian Chemical Society 70 (5), 721–726. Atkins, P., Paula, J., 2006. Physical Chemistry. Oxford Univ. Press, New York. 840 pp. Bergaya, F., Theng, B.K.G., Lagaly, G., 2006. Handbook of Clay Science, Development in Clay Science, vol. 1. Elsevier, Amsterdam. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Cabana, H., Alexandre, C., Agathos, S.N., Jones, J.P., 2009. Immobilization of lactase from the white rot fungus Coriolopsis polyzona and use of the immobilized biocatalyst for the continuous elimination of endocrine disrupting chemicals. Bioresource Technology 100, 3447–3458. Caillou, S., Gerin, P.A., Nonckreman, C.J., Fleith, S., Dupont-Gillain, C.C., Landoulsi, J., Pancera, S.M., Genet, M.J., Rouxhet, P.G., 2008. Enzymes at solid surfaces: nature of the interfaces and physico-chemical processes. Electrochimica Acta 54, 116–122. Carrasco, M.S., Rad, S.C., Gonzalez-Carcedo, 1995. Immobilization of alkaline phosphatase by sorption on Na-sepiolite. Bioresource Technology 51, 175–181. Cetinus, S.A., Öztop, H.N., 2003. Immobilization of catalase into chemically crosslinked chitosan beads. Enzyme and Microbial Technology 32, 889–894. Cetinus, S.A., Şahin, E., Saraydin, D., 2009. Preparation of Cu(II) adsorbed chitosan beads for catalase immobilization. Food Chemistry 114, 962–969. Chang, M.Y., Juang, R.S., 2007. Use of chitosan–clay composite as immobilization support for improved activity and stability of β-glucosidase. Biochemical Engineering Journal 35, 93–98. Choi, M.M.F., Yiu, T.P., 2004. Immobilization of beef liver catalase on eggshell membrane for fabrication of hydrogen peroxide biosensor. Enzyme and Microbial Technology 34, 41–47. Costa, S.A., Tzanov, T., Paar, A., Gudelj, M., Gübitz, G.M., Cavaco-Paulo, A., 2001. Immobilization of catalases from Bacillus SF on alumina for the treatment of textile bleaching effluents. Enzyme and Microbial Technology 28, 815–819. Costa, S.A., Tzanov, T., Carneiro, A.F., Paar, A., Gübitz, G.M., Cavaco-Paulo, A., 2002. Studies of stabilization of native catalase using additives. Enzyme and Microbial Technology 30, 387–391. Demirbas, O., Alkan, M., 2011. Thermodynamics, kinetics and adsorption properties of some biomolecules onto mineral surfaces. In: Piraján, Juan Carlos Moreno (Ed.), Thermodynamics — Kinetics of Dynamic Systems. InTech, pp. 315–330. Domink, L., Lohmann, J., Legge, R.L., 2006. Immobilization of bovine catalase in sol–gels. Enzyme and Microbial Technology 39, 626–633. Eberhardt, A.M., Pedroni, V., Volpe, M., Ferreira, M.L., 2004. Immobilization of catalase from Aspergillus niger on inorganic and biopolymeric supports for H2O2 decomposition. Applied Catalysis B: Environmental 47, 153–163. Grim, R.E., 1968. Clay Mineralogy, 2nd ed. McGraw-Hill, New York. Ho, Y.S., McKay, G., 1999. Pseudo second-order model for sorption processes. Process Biochemistry 34, 451–465. Itoh, T., Ishii, R., Matsuura, S., Hamakawa, S., Hanaoka, T., Tsunoda, T., Mizuguchi, J., Mizukami, F., 2009a. Catalase encapsulated in mesoporous silica and its performance. Biochemical Engineering Journal 44, 167–173. Itoh, T., Ishii, R., Hanaoka, T., Hasegawa, Y., Mizuguchi, J., Shiomib, T., Shimomurab, T., Yamaguchi, A., Kaneda, H., Teramae, N., Mizukami, F., 2009b. Encapsulation of
120
S. Cengiz et al. / Applied Clay Science 65–66 (2012) 114–120
catalase into nanochannels of an inorganic composite membrane. Journal of Molecular Catalysis B: Enzymatic 57, 183–187. Katchalski-Katzir, E., 1993. Immobilized enzymes — learning from past successes and failures. Trends in Biotechnology 11, 471–478. Kilara, A., 1981. Immobilized proteases and lipases. Process 25–27. Krajewska, B., 2009. Ureases. II. Properties and their customizing by enzyme immobilizations: a review. Journal of Molecular Catalysis B: Enzymatic 59, 22–40. Kubal, B.S., D'Souza, S.F., 2004. Immobilization of catalase by entrapment of permeabilized yeast cells in hen egg white using glutaraldehyde. Journal of Biochemical and Biophysical Methods 59, 61–64. Kumar, G.S.V., Kumar, K.S., 2005. Immobilization of catalase on a novel polymer support, crosslinked polystyrene ethylene glycol acrylate resin: role of the macromolecular matrix on enzyme activity. Journal of Applied Polymer Science 97, 8–19. Lagergren, S., 1898. Zur theorie der sogenannten adsorption gelöster stoffe. Kungliga Svenska Vetenskapsakademiens Handlingar 24 (4), 1–39. Li, S., Hu, J., Liu, B., 2004. Use of chemically modified PMMA microspheres for enzyme immobilization. BioSystems 77, 25–32. Moore, D.M., Reynolds Jr., R.C., 1997. X-ray Diffraction and the Identification and Analysis of Minerals. Oxford University Press, New York. Öztürk, N., Tabak, A., Akgöl, S., Denizli, A., 2008. Reversible immobilization of catalase by using a novel bentonite–cysteine (Bent–Cys) microcomposite affinity sorbents. Colloids and Surfaces A: Physicochemical and Engineering Aspects 322, 148–154. Popa, M., Bajan, N., Popa, A.A., Verestiuc, L., 2006. The preparation, characterization and properties of catalase immobilized on crosslinked gellan. Journal of Macromolecular Science, Part A Pure and Applied Chemistry 43, 355–367. Sanjay, G., Sugunan, S., 2006. Enhanced pH and thermal stabilities of invertase immobilized on montmorillonite K-10. Food Chemistry 94, 573–579.
Sedaghat, M.E., Ghiaci, M., Aghaei, H., Soleimanian-Zad, S., 2009. Enzyme immobilization. Part 4. Immobilization of alkaline phosphatase on Na-sepiolite and modified sepiolite. Applied Clay Science 46, 131–135. Shentu, J., Wu, J., Song, W., Jia, Z., 2005. Chitosan microspheres as immobilized dye affinity support for catalase adsorption. International Journal of Biological Macromolecules 37, 42–46. Tarhan, L., 1995. Use of immobilised catalase to remove H2O2 used in the sterilisation of milk. Process Biochemistry 30 (7), 623–628. Tarhan, L., Telefoncu, A., 1992. Effect of enzyme ratio on the properties of glucose oxidase and catalase immobilized on modified perlite. Process Biochemistry 27 (1), 11–15. Tetsuji, I., Ryo, I., Takaaki, H., Yasuhisa, H., Junko, M., Toru, S., Takeshi, S., Akira, Y., Hideaki, K., Norio, T., Fujio, M., 2009. Encapsulation of catalase into nanochannels of an inorganic composite membrane. Journal of Molecular Catalysis B: Enzymatic 57, 183–187. Tukel, S.S., Alptekin, O., 2004. Immobilization and kinetics of catalase onto magnesium silicate. Process Biochemistry 39, 2149–2155. Wang, Q., Xuan Li, C., Fan, X., Wang, P., Cui, L., 2008. Immobilization of catalase on cotton fabric oxidized by sodium periodate. Biocatalysis and Biotransformation 26 (5), 437–443. Ye, P., Xu, Z.K., Che, A.F., Wu, J., Seta, P., 2005. Chitosan-tethered poly (acrylonitrileco-maleic acid) hollow fiber membrane for lipase immobilization. Biomaterials 26, 6394–6403.