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Adsorption, immobilization, and activity of β-glucosidase on different soil colloids
Journal of Colloid and Interface Science 348 (2010) 565–570
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Adsorption, immobilization, and activity of b-glucosidase on different soil colloids Jinlong Yan a,b, Genxing Pan a,*, Lianqing Li a, Guixiang Quan b, Cheng Ding b, Ailan Luo b a b
Institute of Resource, Ecosystem and Environment of Agriculture, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China School of Chemical and Biological Engineering, Yancheng Institute of Technology, 9 Yingbin Avenue, Yancheng 224003, China
a r t i c l e
i n f o
Article history: Received 8 February 2010 Accepted 20 April 2010 Available online 29 April 2010 Keywords: b-Glucosidase Soil colloids Adsorption Immobilization Activity Thermal stability
a b s t r a c t For a better understanding of enzyme stabilization and the subsequent catalytic process in a soil environment, the adsorption, immobilization, and activity of b-glucosidase on various soil colloids from a paddy soil were studied. The calculated parameters maximum adsorption capacity (q0) for fine soil colloids ranged from 169.6 to 203.7 lg mg1, which was higher than coarse soil colloids in the range of 81.0– 94.6 lg mg1, but the lower adsorption affinity (KL) was found on fine soil colloids. The percentages of b-glucosidase desorbed from external surfaces of the coarse soil colloids (27.6–28.5%) were higher than those from the fine soil colloids (17.5–20.2%). b-Glucosidase immobilized on the coarse inorganic and organic soil colloids retained 72.4% and 69.8% of activity, respectively, which indicated the facilitated effect of soil organic matter in the inhibition of enzyme activity. The residual activity for the fine soil clay is 79–81%. After 30 days of storage at 40 °C the free b-glucosidase retained 66.2% of its initial activity, whereas the soil colloidal particle-immobilized enzyme retained 77.1–82.4% of its activity. The half-lives of free b-glucosidase appeared to be 95.9 and 50.4 days at 25 and 40 °C. Immobilization of b-glucosidase on various soil colloids enhanced the thermal stability at all temperatures, and the thermal stability was greatly affected by the affinity between the b-glucosidase molecules and the surface of soil colloidal particles. Due to the protective effect of supports, soil colloidal particle-immobilized enzymes were less sensitive to pH and temperature changes than free enzymes. Data obtained in this study are helpful for further research on the enzymatic mechanisms in carbon cycling and soil carbon storage. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Soil enzymes are constantly being synthesized, accumulated, inactivated, and/or decomposed in the soil, hence playing an important role in catalyzing several important reactions necessary for the life processes of microorganisms in the soil system and the stabilization of soil structure, the decomposition of polymeric material (e.g., cellulose, chitin, and proteins), organic matter formation, and nutrient cycling [1–4]. Burns [5] describes a range of mechanisms by which enzymes accumulate in soil. Free enzymes can be rapidly denatured, degraded, or irreversibly inhibited [6,7]. Therefore, free enzyme activities are normally short-lived. However, soil enzymatic reactions occur in a heterogeneous rather than homogeneous environment [8]. A certain proportion of free enzymes may undergo stabilization through adsorption on soil minerals or through incorporation into humic material [9,10] and, despite affecting their catalytic potential [11], may enable enzyme activity to remain active for long periods in soil. The adsorption and immobilization process can affect the conformational structure of enzymes, and, in turn, enzyme activity and * Corresponding author. Fax: +86 25 84396027. E-mail addresses: [email protected], [email protected] (G. Pan). 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.04.044
kinetic changes may result due to the interaction between enzyme and substrate at solid–solution interfaces [12,13]. Reviews on the accumulation, stabilization mechanisms, and persistence of extracellular enzymes in soils are also given by Kiss et al. [14], Skujins [15], and Ladd et al. [16]. b-Glucosidase is a well-studied enzyme from the glycosidase enzyme family and is important in C cycling. The C content of a soil is significantly correlated to glycosidase enzyme activity, since the enzyme is instrumental in the breakdown of cellulose and is produced by both bacteria and fungi [17]. Some of the recent studies on b-glucosidase focused mainly on the interactions with clay minerals or mineral–organic substance complexes, and included the determination of adsorption, activity, kinetics, and stability [18– 20]. Little information is available on the immobilization of b-glucosidase on various soil colloidal particles. Mechanisms for the immobilization of enzymes on different-sized soil aggregates are still not totally understood. Physical procedures and chemical treatments were used to obtain four kinds of soil colloidal particles from a paddy soil in this research. The objectives of the current work were to investigate the influences of various colloidal particles from a paddy soil on the adsorption, immobilization, and activities of b-glucosidase, and to test the effect of aging, pH, and temperature on the activity of immobilized enzyme. Data obtained
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in this study are helpful for further research on enzymatic mechanisms in carbon cycling and soil carbon storage.
from the initial amount of protein added to the clay. The pellet obtained after the last centrifugation step was stored at 4 °C overnight and utilized for subsequent experiments.
2. Materials and methods 2.4. Desorption studies 2.1. Chemicals b-Glucosidase (EC 3.2.1.21, 8.06 U/mg, Mr = 135,000 from almond) was purchased from Fluka, and p-nitrophenyl-b-D-gulcoside (PNG) was used as the substrate. 2.2. Preparation of soil colloids We used soil from a paddy field in the Wujiang experimental site of Institute of Resources, Ecosystem and Environment of Agriculture, Nanjing Agricultural University at Wujiang Municipality, Suzhou City, Jiangsu Province, China (31°050 2600 N and 120°460 3600 E). According to the World Reference Base for soil resources [21] and the USDA texture classification [22], this soil is a silty clay (Stagnic Anthrosol). The soil was sampled within 0–15 cm of depth and sieved to 2 mm mesh. Two soil colloidal components, i.e., coarse clay (0.2–2 lm) and fine clay (<0.2 lm), were obtained by sedimentation procedures based on Stoke’s law [23]. A part of the fine and coarse clays was treated with 30% hydrogen peroxide to remove the soil organic matter [23]. After saturation with 0.5 mol L1 CaCl2, the colloidal suspension was exhaustively washed with distilled water (until it was free of Cl), and then air-dried. Organic matter content of these soil colloids was determined by the K2CrO4 oxidation technique. Free iron oxide content was determined spectrophotometrically after the extraction by dithionate–citrate–bicarbonate (DCB) [24] and the surface area was measured by the N2 adsorption method. The results are shown in Table 1. 2.3. Adsorption studies Batch experiments were performed in polypropylene tubes with a suspension volume of 10 mL. The suspension of soil colloidal particles (10.0 mg mL1) was dispersed by ultrasonification (3 min at 100 W) in deionized-distilled water (ddH2O). b-Glucosidase solution was prepared in ddH2O at 4 °C (1 mg mL1). Under vigorous stirring, 0.5 mL of the soil suspension was mixed with 1.0 mL of 0.5 mol L1 acetate buffer (pH 5.5) in a 10-mL centrifuge tube. An aliquot of the b-glucosidase solution was added to the tube and the total volume of the suspension in the tube was set at 5.0 mL. The mixture was shaken at 25 °C and 300 rpm until equilibrium was reached (3 h). The suspension was centrifuged at 19,000g and 25 ± 0.5 °C using a Sorvall RC-5B automatic superspeed refrigerated centrifuge. The concentrations of b-glucosidase protein were determined by absorbance at 280 nm (A280) using a digital UV–Vis spectrophotometer from standard curves with the determination limit of 12 lg mL1 and the linearity range of 20– 400 lg mL1. The amount of b-glucosidase protein adsorbed was calculated by subtracting the amount of protein in the supernatant
Table 1 Properties of four kinds of soil colloidal particles prepared from a paddy soil (n = 3).*
*
Soil colloidal particles
Organic matter (%)
Specific surface area (m2 g1)
DCB-Fe (%)
Coarse organic particle Coarse inorganic particle Fine organic particle Fine inorganic particle
1.80 ± 0.16b 0.46 ± 0.09d 2.68 ± 0.24a 0.78 ± 0.12c
204.6 ± 4.6d 229.3 ± 7.4c 263.2 ± 8.2b 298.5 ± 8.8a
4.91 ± 0.24b 5.02 ± 0.25b 8.01 ± 0.43a 8.23 ± 0.43a
In each column varying letters indicate a significant difference (P 6 0.05).
The pellet obtained at an enzyme concentration close to the saturation point was washed thoroughly with 5 mL of 0.1 mol L1 acetate buffer (pH 5.5), using two cycles of resuspension–centrifugation. After this treatment, no further b-glucosidase protein was detected in the supernatant. The supernatant was collected and the concentration of b-glucosidase was measured at 280 nm by spectrophotometry. The percentage desorption from soil colloidal particles and the amount of b-glucosidase immobilized on soil colloidal particles was calculated according to the amount of enzyme bound and desorbed. The pellet obtained after desorption was stored at 4 °C overnight and utilized for subsequent experiments. 2.5. Enzymatic assay A chromogenic assay according to the method of Tabatabai [25] was used to determine the enzymatic activity of free b-glucosidase and b-glucosidase immobilized on the soil colloidal particles. The pellets were suspended in 1 mL of ddH2O and then 0.25 mL of toluene, 4 mL of pH 6.0 modified universal buffer (MUB), and 1 mL of 0.5 mol L1 p-nitrophenyl-b-D-glucoside (PNG) solution were added. The mixtures were incubated at 37 °C for 60 min, and the reaction was stopped by the addition of 1 mL of 0.5 mol L1 CaCl2 and 4 mL of 0.1 mol L1, pH 12, tris(hydroxymethy1)aminomethane (THAM) buffer. Absorbance at 410 nm (A410) was determined with a digital UV–Vis spectrophotometer and compared to a standard curve of p-nitrophenol (Sigma), the product of the enzymatic reaction. The specific activities of free and immobilized enzymes were expressed as microgram p-nitrophenol catalyzed by 1 mg of enzyme within 1 h. The activities of free and immobilized enzymes were measured at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 in the media of MUB. The thermal stability of free and immobilized enzymes was examined by analyzing their activities at elevated temperatures from 17 to 77 °C. To test the effect of aging on the activities of b-glucosidase, the free and immobilized b-glucosidase was stored at constant temperatures of 25 and 40 °C for up to 30 days. All the experiments were conducted in triplicate. Excel 2007 and Origin 7.0 analysis software for windows were used for statistical tests. 3. Results and discussion 3.1. Adsorption of b-glucosidase on various soil colloids Isotherms were the equilibrium relation between the concentrations of the adsorbate in the solid phase and in the liquid phase. The amount of equilibrium adsorption of b-glucosidase on a constant amount of soil colloids particles increased gradually with the increase of b-glucosidase concentration in the solution. Fig. 1 shows that the adsorption curves of b-glucosidase on soil colloids may be classified as L-type isotherm of Giles classification [26]. The L-type isotherm suggested a relatively high affinity between the adsorbate and the adsorbent. A competition may be seen for the sorption sites between water and b-glucosidase. Generally, L-type isotherms reflected the occurrence of chemisorption. Adsorption data were applied to the Langmuir isotherm equation (Fig. 2):
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Table 2 Langmuir parameters for adsorption of b-glucosidase on soil colloidal particles (n = 3). Soil colloidal particles
q0 (lg mg1)*
KL*
r2**
Coarse organic particle Coarse inorganic particle Fine organic particle Fine inorganic particle
94.6 81.0 203.7 169.6
18.74 23.17 11.10 11.65
0.991 0.968 0.996 0.997
*
LSD (P 6 0.05) test showed that the q0 and KL values for adsorption of b-glucosidase on four kinds of soil colloidal particles are significantly different. Significant at 0.001 probability level.
**
Fig. 1. Adsorption isotherms of b-glucosidase on soil colloids. The error bar at each data point represents the standard error (n = 3).
qe ¼ K L C e q0 =ð1 þ K L C e Þ
ð1Þ
1=qe ¼ 1=q0 þ 1=ðK L q0 Þ 1=C e :
ð2Þ
The calculated parameters maximum adsorption capacity (q0, corresponding to complete monolayer coverage) and binding affinity (KL) are listed in Table 2. The results show that the calculated parameters maximum adsorption capacity (q0) for fine soil colloids ranged from 169.6 to 203.7 lg mg1, which was higher than coarse soil colloids in the range of 81.0–94.6 lg mg1. Removal of organic matter with 30% hydrogen peroxide from soil clays resulted in an increase of the surface area for both fine and coarse clays (Table 1). Fine soil particles have larger surface areas and higher contents of organic matter than coarse clays, and considerably greater amounts of iron oxides were also found in the fine clays (Table 1). The higher amount of enzymes adsorbed by fine soil clays has been attributed to the higher content of iron oxides and larger surface areas [23]. It
was assumed that the enzymes were trapped within the macromolecular net of the humic acids and also immobilized at the surface by apparent active adsorption forces [27,28], or adsorbed by soil organic matter in the manner of ion exchange, covalent complexation, and hydrogen bonding [9]. Expandable clays such as smectites have a high affinity for protein adsorption [13]. Staunton and Quiquampoix [29] found that positively charged enzymes (i.e., below their isoelectric points, IEP) were subject to electrostatic interactions with clay minerals. On the other hand, Geiger et al. [30] reported that there was no inhibition of b-glucosidase activity by goethite even when about 95% of the enzyme was absorbed on the surfaces of the oxide, and concluded that the adsorption was caused principally by nonelectrostatic forces which were too weak to affect the structure of the enzyme. Goethite is the most widespread iron oxide in soils [31]; its chemical nature and the large specific surface area make it an efficient sorbent for protein molecules. Safari Sinegani et al. [32] found that organic matters adsorbed and immobilized cellulase protein more than clay minerals; a large part of the cellulolytic activities of soil depends on the enzymes immobilized on its organic matters, especially those with high C/N ratios. The lower adsorption affinity was found on fine soil colloids. The KL value reflected the binding energy between the b-glucosidase molecules and the surface of soil colloidal particles; the greater the KL value the higher the affinity. Huang et al. attributed the lower affinity of fine clays for enzymes to their higher contents of iron oxides [23]. 3.2. Desorption of absorbed b-glucosidase from soil colloidal particles Desorption of absorbed b-glucosidase from soil colloidal particles by 0.1 mol L1 acetate buffers (pH 5.5) is shown in Fig. 3.
Fig. 2. Langmuir linear isotherms of b-glucosidase on soil colloids. The error bar at each data point represents the standard error (n = 3).
Fig. 3. Desorption ratios of b-glucosidase from absorbed soil colloids. The error bar at each data point represents the standard error (n = 3).
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Results show that a large proportion of enzyme molecules were immobilized on soil colloidal particle clays after the washing of acetate buffers, indicating that the interaction of enzyme molecules with soil colloidal particles may be chemical and van der Waals force and/or hydrogen bonding may play important roles in the adsorption of enzyme. As an excellent hydrogen-bond donor for the protonated amino group (—NHþ 3 ), a hydrogen bond can form with the structural oxygen of siloxane surfaces [33]. The percentages of b-glucosidase desorbed from surfaces of the coarse soil colloids (27.6–28.5%) were higher than those from the fine soil colloids (17.5–20.2%). It was evident that in comparison to fine clays more loosely bound enzymes were attached on coarse soil clays, meaning that enzymes adsorbed on soil organic components can be easily removed. In a study of the influence of electrostatic forces on protein adsorption [34], atomic force microscopy (AFM) was used to measure the electrostatic forces on the sorption of human serum albumin by UV-ozone-modified polystyrene. Results show that the electrostatic double force, introduced in an electrolyte near a charged surface, is an important driving factor (attractive or repulsive) for the process of protein adsorption onto a surface. The process of the spatial adsorption of proteins onto surfaces may be precisely controlled by the design of the electrostatic field profile near the surface and by the adjustment of the protein isoelectric point. Our research also revealed that more enzyme molecules were adsorbed on organic fractions, although this difference was generally small. As only a small number of soil colloidal particles were studied, no conclusions can be reached about which soil colloid properties caused this variation, although the electrostatic force between enzymes and soil colloids appears to be important. No remarkable difference was observed for the amount of immobilized b-glucosidase on inorganic and organic particles of the same size due to the larger desorption ratio of organic particles. 3.3. Activity of immobilized b-glucosidase b-Glucosidase immobilized on the coarse inorganic and organic soil colloidal particles retained 72.4% and 69.8% of activity, respectively (1 day of aging at 40 °C in Fig. 4b). The residual activity for the fine soil clay is 79–81%. Hence, the decrease in the specific activity of b-glucosidase on immobilization indicated that some of the enzyme active sites were hindered by the soil colloidal
particle surfaces [33], probably as a result of the inaccessibility or modifications of the active site of the enzymes. The results suggested that enzymes immobilized on fine soil particle retained higher activities. Gianfreda et al. illustrated that the formation of enzyme–iron complexes may enhance its binding with the substrate and resulted in higher enzymatic activity [35]. Enzyme activity was also inhibited in the presence of organic matter. Several investigators [36,37] reported on the synthetic and natural humic compounds on enzyme activity previously. Mechanisms of the inhibitory action of soil organic matter were also proposed, such as the conformational change of the enzyme due to complexation of the active sites in the enzyme molecules by humic acids, the binding of the substrate to humic acids, and the competition effect of humic compounds with substrate for the catalytically active site [36]. 3.4. Influence of aging on activities of free and immobilized b-glucosidase The stability of the immobilized b-glucosidase was compared with that of the free enzyme at 25 and 40 °C. When stored for 11 days at 25 °C the activity of the immobilized b-glucosidase was preserved at 100%, whereas the activity of the free enzyme drastically decreased (Fig. 4a). After 30 days of storage at 25 °C the free b-glucosidase retained 81.4% of its initial activity whereas the soil fine coarse particle-immobilized b-glucosidase retained 93.7% of its activity. After 30 days of storage at 40 °C the free b-glucosidase retained 66.2% of its initial activity, whereas the soil colloidal particle-immobilized enzyme retained 77.1–82.4% of its activity (Fig. 4b). These results clearly indicated that the immobilized b-glucosidase on soil colloidal particles was more stable and less sensitive to the aging process than the free b-glucosidase. Naidja et al. [33] showed that whether stored at 4 or 25 °C for 30 days immobilized tyrosinase on Al(OH)3–montmorillonite complex at a level of coatings of 5.0 mmol Al/g clay was more stable than free tyrosinase. A linear regression analysis method was used for the further study of the thermal properties [38]. The semilogarithmic plots of the remaining percentage b-glucosidase activity vs. time at 25 and 40 °C are shown in Fig. 5. The thermal stabilities of free and immobilized b-glucosidase in terms of their half-life were calculated from the regression analysis plots at different temperatures. As shown in Table 3, the half-lives of free b-glucosidase appeared
Fig. 4. Influence of aging at 25 °C (a) to 40 °C (b) on activities of free and immobilized b-glucosidase. The error bar at each data point represents the standard error (n = 3).
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J. Yan et al. / Journal of Colloid and Interface Science 348 (2010) 565–570 Table 3 Half-lives of free and immobilized b-glucosidase at 25 and 40 °C (n = 3). Enzyme
25 °C Half-life (days)
Free Immobilized on coarse organic particle Immobilized on coarse inorganic particle Immobilized on fine organic particle Immobilized on fine inorganic particle
40 °C *
r
2**
Half-life (days)*
r2**
95.9 347.2
0.994 0.982
50.4 102.7
0.981 0.987
286.8
0.976
94.1
0.994
299.0
0.980
76.4
0.988
266.7
0.978
80.6
0.993
* LSD (P 6 0.05) test showed that the half-lives of free b-glucosidase are significantly different from the soil colloidal particle-immobilized enzymes at the same aging temperature. ** Significant at 0.001 probability level.
to be 95.9 and 50.4 days at 25 and 40 °C, respectively. Immobilization of b-glucosidase on various soil colloids enhanced the thermal stability at all temperatures (Table 3). In this case, the r2 correlation coefficients were higher than 0.98, with 95% confidence, despite some important deviation between the model prediction and the experimental half-lives. Results supported that the thermal stability of free b-glucosidase drastically decreased with temperature and can be rapidly denatured. Coarse soil colloid-immobilized b-glucosidases presented the higher thermal stability than fine colloids (Table 3). It probably meant that the thermal stability of the immobilized enzyme was greatly affected by the affinity between the b-glucosidase molecules and the surface of soil colloidal particles, the higher affinity with the higher thermal stability. This finding may be useful in guiding and designing enzyme immobilization engineering, and helpful for further research on enzymatic mechanisms in carbon cycling and soil carbon storage. Usually, the soil colloidal support has a protecting effect from heat when the immobilized enzyme inactivation occurs. The immobilization step affected the conformational flexibility and caused an increase in enzyme rigidity, which was commonly reflected by an increase in stability toward denaturation by raising the temperature [39]. 3.5. Effect of pH on activities of free and immobilized b-glucosidase Fig. 5. Semilogarithmic plots of remaining free and immobilized enzyme as a function of time of incubation at 25 °C (a) and 40 °C (b). The error bar at each data point represents the standard error (n = 3).
A similar pH–activity profile was observed for the free and immobilized enzyme in the range of pH 4.5–7.0 in Fig. 6a. Both free
Fig. 6. Effects of pH (a) and temperature (b) on activities of free and immobilized b-glucosidase. The error bar at each data point represents the standard error (n = 3).
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and immobilized b-glucosidase displayed the highest activity at pH 6.0. No obvious shift for the optimal pH of immobilized enzyme was observed as described previously by some investigators [38]. However, the decline of activity as pH increased from 6.0 to 7.0 or decreased from 6.0 to 4.0 was much more sharp for free b-glucosidase than immobilized b-glucosidase. Instead, more flat curves were observed for b-glucosidase immobilized on soil colloidal particles. These results suggested that free enzyme was more sensitive to pH changes than soil colloidal particle-immobilized enzymes. 3.6. Effect of temperature on activities of free and immobilized b-glucosidase The role of the immobilization on the catalytic behavior of b-glucosidase was evident by temperature–activity curves illustrated in Fig. 6b at temperatures from 17 to 77 °C. The figure clearly shows that both free and immobilized enzymes had an optimal activity at 57 °C. However, a smooth behavior (i.e., less dependence on temperature) was in general exhibited by b-glucosidase immobilized on soil colloidal particles, which indicated that the thermal stability of the immobilized b-glucosidase was higher than the free enzyme. As temperature increased from 57 to 77 °C, the activity of free b-glucosidase decreased by 31.2%; immobilized enzymes declined by 8.2–15.3%. Results suggested that the thermal stability of immobilized b-glucosidase becomes significantly higher than that of free b-glucosidase at higher temperatures. Immobilization of b-glucosidase on various soil colloids from a paddy soil is supposed to preserve the tertiary structure enzyme, and it protected the enzyme from conformational changes causing effects of the environment. The results also revealed that temperature–activity profiles of coarse colloid-immobilized enzymes were more flat than fine clays. For the same particle size of clay fractions, the thermal stability for organic clays was higher than that for inorganic clays. Similar results were reported by Rao et al. [40]. 4. Summary Higher adsorption amounts and lower adsorption affinity of b-glucosidase were found on fine soil colloidal particles, which were attributed to their higher surface area and higher content of iron oxides. b-Glucosidase adsorbed on coarse soil colloidal particles was also more easily released. More enzyme molecules were adsorbed on organic soil colloidal particles, confirming the facilitated effect of organic substances in the adsorption of enzymes. Decrease in the specific activity of b-glucosidase on immobilization indicated that some of the enzyme active sites were hindered by the soil colloidal particle surfaces, probably as the result of the inaccessibility or modifications of the active site of the enzymes. Immobilized b-glucosidase on soil colloidal particles was more stable and less sensitive to the aging process than the free b-glucosidase. In terms of their half-life, immobilization of b-glucosidase on various soil colloids enhanced the thermal stability at all temperature. Due to the protective effect of supports, soil colloidal particleimmobilized enzymes were also less sensitive to pH and temperature changes than free enzyme. Data obtained in this study are helpful for further research on enzymatic mechanisms in carbon cycling and soil carbon storage.
Acknowledgments This study was supported by Major Program of National Natural Science Foundation of China (No. 40830528) and the Natural Science Foundation of Department of Education, Jiangsu Province (08KJB210002). References [1] C. Trasar-Cepeda, F. Gil-Sotres, M.C. Leirós, Soil Biol. Biochem. 39 (2007) 311. [2] R.P. Dick, in: J.W. Doran, D.C. Coleman, D.F. Bezdicek, B.A. Stewart (Eds.), Defining Soil Quality for a Substainable Environment, Soil Science Society of America, Madison, WI, 1994. [3] C. Trasar-Cepeda, M.C. Leirós, F. Gil-Sotres, Soil Biol. Biochem. 40 (2008) 2146. [4] P. Nannipieri, E. Kandeler, P. Ruggiero, in: R.G. Burns, R.P. Dick (Eds.), Enzymes in the Environment, Dekker, New York, 2002. [5] R.G. Burns, Soil Biol. Biochem. 14 (1982) 423. [6] M.-C. Marx, E. Kandeler, M. Wood, N. Wermbter, S.C. Jarvis, Soil Biol. Biochem. 37 (2005) 35. [7] J.M. Sarkar, A. Leonowicz, J.-M. Bollag, Soil Biol. Biochem. 21 (1989) 223. [8] P. Nannipieri, L. Gianfreda, in: P.M. Huang, N. Senesi, J. Buffle (Eds.), Environmental Particles Structure and Surface Reactions of Soil Particles, Wiley, New York, 1998. [9] P. Nannipieri, P. Sequi, P. Fusi, in: A. Piccolo (Ed.), Humic Substances in Terrestrial Ecosystems, Elsevier, Amsterdam, 1996. [10] R.G. Burns, in: P.M. Huang, M. Schnitzer (Eds.), Interactions of Soil Minerals with Natural Organics and Microbes, Soil Science Society of America, Madison, WI, 1986. [11] M.D. Busto, M. Perez-Mateos, Eur. J. Soil Sci. 51 (2000) 193. [12] H. Quiquampoix, Biochimie 69 (1987) 753. [13] A.A. Safari-Sinegani, G. Emtiazi, H. Shariatmadari, J. Colloid Interface Sci. 290 (2005) 39. [14] S. Kiss, M. Draga-Bularda, D. Radulescu, Adv. Agron. 27 (1975) 25. [15] J. Skujins, CRC Crit. Rev. Microbiol. 4 (1976) 383. [16] J.N. Ladd, R.C. Foster, P. Nannipieri, J.M. Oades, in: G. Stotzky, J. Bollag (Eds.), Soil Biochemistry, vol. 9, Dekker, New York, 1996. [17] S.P. Deng, M.A. Tabatabai, Biol. Fertil. Soils 22 (1996) 208. [18] H. Quiquampoix, Biochimie 69 (1987) 765. [19] G. Geiger, H. Brandl, G. Furrer, R. Schulin, Soil Biol. Biochem. 30 (1998) 1537. [20] M.-Y. Chang, R.-S. Juang, Biochem. Eng. J. 35 (2007) 93. [21] IUSS Working Group WRB, World Soil Resources Reports No. 103, FAO, Rome, 2006. [22] Soil Survey Staff, Soil Survey Investigations Report No. 45, Version 1.0, USDANRCS, National Soil Survey Center, Lincoln, NE, 1995. [23] Q. Huang, W. Liang, P. Cai, Colloids Surf. B 45 (2005) 209. [24] Y. Xiong, Soil Colloids, vol. 2, Science Press, Beijing, 1983 (in Chinese). [25] M.A. Tabatabai, in: R.W. Weaver, J.S. Angel, P.S. Bottomley (Eds.), Methods of Soil Analysis, Part 2, Microbiological and Biochemical Properties, Soil Science Society of America, Madison, WI, 1994. [26] C.H. Giles, T.H. MacEwan, S.N. Nakhwa, D. Smith, J. Chem. Soc. 56 (1960) 3973. [27] S.A. Boyd, M.M. Mortland, in: J.-M. Bollag, G. Stotzky (Eds.), Soil Biochemistry, Dekker, 1990. [28] P. Ruggiero, J. Dec, J.-M. Bollag, in: G. Stotzky, J.-M. Bollag (Eds.), Soil Biochemistry, vol. 9, Dekker, New York, 1996. [29] S. Staunton, H. Quiquampoix, J. Colloid Interface Sci. 166 (1994) 89. [30] G. Geiger, M.P. Livinston, F. Funk, R. Schulin, Eur. J. Soil Sci. 49 (1998) 17. [31] P.G. Grossi, D.L. Sparks, C.C. Ainsworth, Environ. Sci. Technol. 28 (1994) 1422. [32] A.A. Safari Sinegani, G. Emtiazi, H. Shariatmadari, in: A. Faz, R. Ortiz, A.R. Mermut (Eds.), International Symposium on Sustainable Use and Management of Soils in Arid and Semiarid Regions, vol. II, Cartagena, Murcia, Spain, 2002. [33] A. Naidja, P.M. Huang, J.-M. Bollag, J. Mol. Catal. A: Chem. 115 (1997) 305. [34] G.V. Lubarsky, M.M. Browne, S.A. Mitchell, M.R. Davidson, R.H. Bradley, Colloids Surf. B 44 (2005) 56. [35] L. Gianfreda, M.A. Rao, A. Violante, Soil Sci. Soc. Am. J. 59 (1995) 805. [36] L. Gianfreda, M.A. Rao, A. Violante, Soil Biol. Biochem. 23 (1991) 581. [37] W.A. Dick, N.G. Juma, M.A. Tabatabai, Soil Sci. 136 (1983) 19. [38] S.K. Srivastava, K.S. Gopalkrishnan, K.B. Ramachandran, Enzyme Microbiol. Technol. 6 (1984) 508. [39] M.A. Abdel-Naby, Appl. Biochem. Biotechnol. 38 (1993) 69. [40] M.A. Rao, A. Violante, L. Gianfreda, Soil Biol. Biochem. 32 (2000) 1007.
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Adsorption, immobilization, and activity of b-glucosidase on different soil colloids Jinlong Yan a,b, Genxing Pan a,*, Lianqing Li a, Guixiang Quan b, Cheng Ding b, Ailan Luo b a b
Institute of Resource, Ecosystem and Environment of Agriculture, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China School of Chemical and Biological Engineering, Yancheng Institute of Technology, 9 Yingbin Avenue, Yancheng 224003, China
a r t i c l e
i n f o
Article history: Received 8 February 2010 Accepted 20 April 2010 Available online 29 April 2010 Keywords: b-Glucosidase Soil colloids Adsorption Immobilization Activity Thermal stability
a b s t r a c t For a better understanding of enzyme stabilization and the subsequent catalytic process in a soil environment, the adsorption, immobilization, and activity of b-glucosidase on various soil colloids from a paddy soil were studied. The calculated parameters maximum adsorption capacity (q0) for fine soil colloids ranged from 169.6 to 203.7 lg mg1, which was higher than coarse soil colloids in the range of 81.0– 94.6 lg mg1, but the lower adsorption affinity (KL) was found on fine soil colloids. The percentages of b-glucosidase desorbed from external surfaces of the coarse soil colloids (27.6–28.5%) were higher than those from the fine soil colloids (17.5–20.2%). b-Glucosidase immobilized on the coarse inorganic and organic soil colloids retained 72.4% and 69.8% of activity, respectively, which indicated the facilitated effect of soil organic matter in the inhibition of enzyme activity. The residual activity for the fine soil clay is 79–81%. After 30 days of storage at 40 °C the free b-glucosidase retained 66.2% of its initial activity, whereas the soil colloidal particle-immobilized enzyme retained 77.1–82.4% of its activity. The half-lives of free b-glucosidase appeared to be 95.9 and 50.4 days at 25 and 40 °C. Immobilization of b-glucosidase on various soil colloids enhanced the thermal stability at all temperatures, and the thermal stability was greatly affected by the affinity between the b-glucosidase molecules and the surface of soil colloidal particles. Due to the protective effect of supports, soil colloidal particle-immobilized enzymes were less sensitive to pH and temperature changes than free enzymes. Data obtained in this study are helpful for further research on the enzymatic mechanisms in carbon cycling and soil carbon storage. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Soil enzymes are constantly being synthesized, accumulated, inactivated, and/or decomposed in the soil, hence playing an important role in catalyzing several important reactions necessary for the life processes of microorganisms in the soil system and the stabilization of soil structure, the decomposition of polymeric material (e.g., cellulose, chitin, and proteins), organic matter formation, and nutrient cycling [1–4]. Burns [5] describes a range of mechanisms by which enzymes accumulate in soil. Free enzymes can be rapidly denatured, degraded, or irreversibly inhibited [6,7]. Therefore, free enzyme activities are normally short-lived. However, soil enzymatic reactions occur in a heterogeneous rather than homogeneous environment [8]. A certain proportion of free enzymes may undergo stabilization through adsorption on soil minerals or through incorporation into humic material [9,10] and, despite affecting their catalytic potential [11], may enable enzyme activity to remain active for long periods in soil. The adsorption and immobilization process can affect the conformational structure of enzymes, and, in turn, enzyme activity and * Corresponding author. Fax: +86 25 84396027. E-mail addresses: [email protected], [email protected] (G. Pan). 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.04.044
kinetic changes may result due to the interaction between enzyme and substrate at solid–solution interfaces [12,13]. Reviews on the accumulation, stabilization mechanisms, and persistence of extracellular enzymes in soils are also given by Kiss et al. [14], Skujins [15], and Ladd et al. [16]. b-Glucosidase is a well-studied enzyme from the glycosidase enzyme family and is important in C cycling. The C content of a soil is significantly correlated to glycosidase enzyme activity, since the enzyme is instrumental in the breakdown of cellulose and is produced by both bacteria and fungi [17]. Some of the recent studies on b-glucosidase focused mainly on the interactions with clay minerals or mineral–organic substance complexes, and included the determination of adsorption, activity, kinetics, and stability [18– 20]. Little information is available on the immobilization of b-glucosidase on various soil colloidal particles. Mechanisms for the immobilization of enzymes on different-sized soil aggregates are still not totally understood. Physical procedures and chemical treatments were used to obtain four kinds of soil colloidal particles from a paddy soil in this research. The objectives of the current work were to investigate the influences of various colloidal particles from a paddy soil on the adsorption, immobilization, and activities of b-glucosidase, and to test the effect of aging, pH, and temperature on the activity of immobilized enzyme. Data obtained
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in this study are helpful for further research on enzymatic mechanisms in carbon cycling and soil carbon storage.
from the initial amount of protein added to the clay. The pellet obtained after the last centrifugation step was stored at 4 °C overnight and utilized for subsequent experiments.
2. Materials and methods 2.4. Desorption studies 2.1. Chemicals b-Glucosidase (EC 3.2.1.21, 8.06 U/mg, Mr = 135,000 from almond) was purchased from Fluka, and p-nitrophenyl-b-D-gulcoside (PNG) was used as the substrate. 2.2. Preparation of soil colloids We used soil from a paddy field in the Wujiang experimental site of Institute of Resources, Ecosystem and Environment of Agriculture, Nanjing Agricultural University at Wujiang Municipality, Suzhou City, Jiangsu Province, China (31°050 2600 N and 120°460 3600 E). According to the World Reference Base for soil resources [21] and the USDA texture classification [22], this soil is a silty clay (Stagnic Anthrosol). The soil was sampled within 0–15 cm of depth and sieved to 2 mm mesh. Two soil colloidal components, i.e., coarse clay (0.2–2 lm) and fine clay (<0.2 lm), were obtained by sedimentation procedures based on Stoke’s law [23]. A part of the fine and coarse clays was treated with 30% hydrogen peroxide to remove the soil organic matter [23]. After saturation with 0.5 mol L1 CaCl2, the colloidal suspension was exhaustively washed with distilled water (until it was free of Cl), and then air-dried. Organic matter content of these soil colloids was determined by the K2CrO4 oxidation technique. Free iron oxide content was determined spectrophotometrically after the extraction by dithionate–citrate–bicarbonate (DCB) [24] and the surface area was measured by the N2 adsorption method. The results are shown in Table 1. 2.3. Adsorption studies Batch experiments were performed in polypropylene tubes with a suspension volume of 10 mL. The suspension of soil colloidal particles (10.0 mg mL1) was dispersed by ultrasonification (3 min at 100 W) in deionized-distilled water (ddH2O). b-Glucosidase solution was prepared in ddH2O at 4 °C (1 mg mL1). Under vigorous stirring, 0.5 mL of the soil suspension was mixed with 1.0 mL of 0.5 mol L1 acetate buffer (pH 5.5) in a 10-mL centrifuge tube. An aliquot of the b-glucosidase solution was added to the tube and the total volume of the suspension in the tube was set at 5.0 mL. The mixture was shaken at 25 °C and 300 rpm until equilibrium was reached (3 h). The suspension was centrifuged at 19,000g and 25 ± 0.5 °C using a Sorvall RC-5B automatic superspeed refrigerated centrifuge. The concentrations of b-glucosidase protein were determined by absorbance at 280 nm (A280) using a digital UV–Vis spectrophotometer from standard curves with the determination limit of 12 lg mL1 and the linearity range of 20– 400 lg mL1. The amount of b-glucosidase protein adsorbed was calculated by subtracting the amount of protein in the supernatant
Table 1 Properties of four kinds of soil colloidal particles prepared from a paddy soil (n = 3).*
*
Soil colloidal particles
Organic matter (%)
Specific surface area (m2 g1)
DCB-Fe (%)
Coarse organic particle Coarse inorganic particle Fine organic particle Fine inorganic particle
1.80 ± 0.16b 0.46 ± 0.09d 2.68 ± 0.24a 0.78 ± 0.12c
204.6 ± 4.6d 229.3 ± 7.4c 263.2 ± 8.2b 298.5 ± 8.8a
4.91 ± 0.24b 5.02 ± 0.25b 8.01 ± 0.43a 8.23 ± 0.43a
In each column varying letters indicate a significant difference (P 6 0.05).
The pellet obtained at an enzyme concentration close to the saturation point was washed thoroughly with 5 mL of 0.1 mol L1 acetate buffer (pH 5.5), using two cycles of resuspension–centrifugation. After this treatment, no further b-glucosidase protein was detected in the supernatant. The supernatant was collected and the concentration of b-glucosidase was measured at 280 nm by spectrophotometry. The percentage desorption from soil colloidal particles and the amount of b-glucosidase immobilized on soil colloidal particles was calculated according to the amount of enzyme bound and desorbed. The pellet obtained after desorption was stored at 4 °C overnight and utilized for subsequent experiments. 2.5. Enzymatic assay A chromogenic assay according to the method of Tabatabai [25] was used to determine the enzymatic activity of free b-glucosidase and b-glucosidase immobilized on the soil colloidal particles. The pellets were suspended in 1 mL of ddH2O and then 0.25 mL of toluene, 4 mL of pH 6.0 modified universal buffer (MUB), and 1 mL of 0.5 mol L1 p-nitrophenyl-b-D-glucoside (PNG) solution were added. The mixtures were incubated at 37 °C for 60 min, and the reaction was stopped by the addition of 1 mL of 0.5 mol L1 CaCl2 and 4 mL of 0.1 mol L1, pH 12, tris(hydroxymethy1)aminomethane (THAM) buffer. Absorbance at 410 nm (A410) was determined with a digital UV–Vis spectrophotometer and compared to a standard curve of p-nitrophenol (Sigma), the product of the enzymatic reaction. The specific activities of free and immobilized enzymes were expressed as microgram p-nitrophenol catalyzed by 1 mg of enzyme within 1 h. The activities of free and immobilized enzymes were measured at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 in the media of MUB. The thermal stability of free and immobilized enzymes was examined by analyzing their activities at elevated temperatures from 17 to 77 °C. To test the effect of aging on the activities of b-glucosidase, the free and immobilized b-glucosidase was stored at constant temperatures of 25 and 40 °C for up to 30 days. All the experiments were conducted in triplicate. Excel 2007 and Origin 7.0 analysis software for windows were used for statistical tests. 3. Results and discussion 3.1. Adsorption of b-glucosidase on various soil colloids Isotherms were the equilibrium relation between the concentrations of the adsorbate in the solid phase and in the liquid phase. The amount of equilibrium adsorption of b-glucosidase on a constant amount of soil colloids particles increased gradually with the increase of b-glucosidase concentration in the solution. Fig. 1 shows that the adsorption curves of b-glucosidase on soil colloids may be classified as L-type isotherm of Giles classification [26]. The L-type isotherm suggested a relatively high affinity between the adsorbate and the adsorbent. A competition may be seen for the sorption sites between water and b-glucosidase. Generally, L-type isotherms reflected the occurrence of chemisorption. Adsorption data were applied to the Langmuir isotherm equation (Fig. 2):
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Table 2 Langmuir parameters for adsorption of b-glucosidase on soil colloidal particles (n = 3). Soil colloidal particles
q0 (lg mg1)*
KL*
r2**
Coarse organic particle Coarse inorganic particle Fine organic particle Fine inorganic particle
94.6 81.0 203.7 169.6
18.74 23.17 11.10 11.65
0.991 0.968 0.996 0.997
*
LSD (P 6 0.05) test showed that the q0 and KL values for adsorption of b-glucosidase on four kinds of soil colloidal particles are significantly different. Significant at 0.001 probability level.
**
Fig. 1. Adsorption isotherms of b-glucosidase on soil colloids. The error bar at each data point represents the standard error (n = 3).
qe ¼ K L C e q0 =ð1 þ K L C e Þ
ð1Þ
1=qe ¼ 1=q0 þ 1=ðK L q0 Þ 1=C e :
ð2Þ
The calculated parameters maximum adsorption capacity (q0, corresponding to complete monolayer coverage) and binding affinity (KL) are listed in Table 2. The results show that the calculated parameters maximum adsorption capacity (q0) for fine soil colloids ranged from 169.6 to 203.7 lg mg1, which was higher than coarse soil colloids in the range of 81.0–94.6 lg mg1. Removal of organic matter with 30% hydrogen peroxide from soil clays resulted in an increase of the surface area for both fine and coarse clays (Table 1). Fine soil particles have larger surface areas and higher contents of organic matter than coarse clays, and considerably greater amounts of iron oxides were also found in the fine clays (Table 1). The higher amount of enzymes adsorbed by fine soil clays has been attributed to the higher content of iron oxides and larger surface areas [23]. It
was assumed that the enzymes were trapped within the macromolecular net of the humic acids and also immobilized at the surface by apparent active adsorption forces [27,28], or adsorbed by soil organic matter in the manner of ion exchange, covalent complexation, and hydrogen bonding [9]. Expandable clays such as smectites have a high affinity for protein adsorption [13]. Staunton and Quiquampoix [29] found that positively charged enzymes (i.e., below their isoelectric points, IEP) were subject to electrostatic interactions with clay minerals. On the other hand, Geiger et al. [30] reported that there was no inhibition of b-glucosidase activity by goethite even when about 95% of the enzyme was absorbed on the surfaces of the oxide, and concluded that the adsorption was caused principally by nonelectrostatic forces which were too weak to affect the structure of the enzyme. Goethite is the most widespread iron oxide in soils [31]; its chemical nature and the large specific surface area make it an efficient sorbent for protein molecules. Safari Sinegani et al. [32] found that organic matters adsorbed and immobilized cellulase protein more than clay minerals; a large part of the cellulolytic activities of soil depends on the enzymes immobilized on its organic matters, especially those with high C/N ratios. The lower adsorption affinity was found on fine soil colloids. The KL value reflected the binding energy between the b-glucosidase molecules and the surface of soil colloidal particles; the greater the KL value the higher the affinity. Huang et al. attributed the lower affinity of fine clays for enzymes to their higher contents of iron oxides [23]. 3.2. Desorption of absorbed b-glucosidase from soil colloidal particles Desorption of absorbed b-glucosidase from soil colloidal particles by 0.1 mol L1 acetate buffers (pH 5.5) is shown in Fig. 3.
Fig. 2. Langmuir linear isotherms of b-glucosidase on soil colloids. The error bar at each data point represents the standard error (n = 3).
Fig. 3. Desorption ratios of b-glucosidase from absorbed soil colloids. The error bar at each data point represents the standard error (n = 3).
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Results show that a large proportion of enzyme molecules were immobilized on soil colloidal particle clays after the washing of acetate buffers, indicating that the interaction of enzyme molecules with soil colloidal particles may be chemical and van der Waals force and/or hydrogen bonding may play important roles in the adsorption of enzyme. As an excellent hydrogen-bond donor for the protonated amino group (—NHþ 3 ), a hydrogen bond can form with the structural oxygen of siloxane surfaces [33]. The percentages of b-glucosidase desorbed from surfaces of the coarse soil colloids (27.6–28.5%) were higher than those from the fine soil colloids (17.5–20.2%). It was evident that in comparison to fine clays more loosely bound enzymes were attached on coarse soil clays, meaning that enzymes adsorbed on soil organic components can be easily removed. In a study of the influence of electrostatic forces on protein adsorption [34], atomic force microscopy (AFM) was used to measure the electrostatic forces on the sorption of human serum albumin by UV-ozone-modified polystyrene. Results show that the electrostatic double force, introduced in an electrolyte near a charged surface, is an important driving factor (attractive or repulsive) for the process of protein adsorption onto a surface. The process of the spatial adsorption of proteins onto surfaces may be precisely controlled by the design of the electrostatic field profile near the surface and by the adjustment of the protein isoelectric point. Our research also revealed that more enzyme molecules were adsorbed on organic fractions, although this difference was generally small. As only a small number of soil colloidal particles were studied, no conclusions can be reached about which soil colloid properties caused this variation, although the electrostatic force between enzymes and soil colloids appears to be important. No remarkable difference was observed for the amount of immobilized b-glucosidase on inorganic and organic particles of the same size due to the larger desorption ratio of organic particles. 3.3. Activity of immobilized b-glucosidase b-Glucosidase immobilized on the coarse inorganic and organic soil colloidal particles retained 72.4% and 69.8% of activity, respectively (1 day of aging at 40 °C in Fig. 4b). The residual activity for the fine soil clay is 79–81%. Hence, the decrease in the specific activity of b-glucosidase on immobilization indicated that some of the enzyme active sites were hindered by the soil colloidal
particle surfaces [33], probably as a result of the inaccessibility or modifications of the active site of the enzymes. The results suggested that enzymes immobilized on fine soil particle retained higher activities. Gianfreda et al. illustrated that the formation of enzyme–iron complexes may enhance its binding with the substrate and resulted in higher enzymatic activity [35]. Enzyme activity was also inhibited in the presence of organic matter. Several investigators [36,37] reported on the synthetic and natural humic compounds on enzyme activity previously. Mechanisms of the inhibitory action of soil organic matter were also proposed, such as the conformational change of the enzyme due to complexation of the active sites in the enzyme molecules by humic acids, the binding of the substrate to humic acids, and the competition effect of humic compounds with substrate for the catalytically active site [36]. 3.4. Influence of aging on activities of free and immobilized b-glucosidase The stability of the immobilized b-glucosidase was compared with that of the free enzyme at 25 and 40 °C. When stored for 11 days at 25 °C the activity of the immobilized b-glucosidase was preserved at 100%, whereas the activity of the free enzyme drastically decreased (Fig. 4a). After 30 days of storage at 25 °C the free b-glucosidase retained 81.4% of its initial activity whereas the soil fine coarse particle-immobilized b-glucosidase retained 93.7% of its activity. After 30 days of storage at 40 °C the free b-glucosidase retained 66.2% of its initial activity, whereas the soil colloidal particle-immobilized enzyme retained 77.1–82.4% of its activity (Fig. 4b). These results clearly indicated that the immobilized b-glucosidase on soil colloidal particles was more stable and less sensitive to the aging process than the free b-glucosidase. Naidja et al. [33] showed that whether stored at 4 or 25 °C for 30 days immobilized tyrosinase on Al(OH)3–montmorillonite complex at a level of coatings of 5.0 mmol Al/g clay was more stable than free tyrosinase. A linear regression analysis method was used for the further study of the thermal properties [38]. The semilogarithmic plots of the remaining percentage b-glucosidase activity vs. time at 25 and 40 °C are shown in Fig. 5. The thermal stabilities of free and immobilized b-glucosidase in terms of their half-life were calculated from the regression analysis plots at different temperatures. As shown in Table 3, the half-lives of free b-glucosidase appeared
Fig. 4. Influence of aging at 25 °C (a) to 40 °C (b) on activities of free and immobilized b-glucosidase. The error bar at each data point represents the standard error (n = 3).
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J. Yan et al. / Journal of Colloid and Interface Science 348 (2010) 565–570 Table 3 Half-lives of free and immobilized b-glucosidase at 25 and 40 °C (n = 3). Enzyme
25 °C Half-life (days)
Free Immobilized on coarse organic particle Immobilized on coarse inorganic particle Immobilized on fine organic particle Immobilized on fine inorganic particle
40 °C *
r
2**
Half-life (days)*
r2**
95.9 347.2
0.994 0.982
50.4 102.7
0.981 0.987
286.8
0.976
94.1
0.994
299.0
0.980
76.4
0.988
266.7
0.978
80.6
0.993
* LSD (P 6 0.05) test showed that the half-lives of free b-glucosidase are significantly different from the soil colloidal particle-immobilized enzymes at the same aging temperature. ** Significant at 0.001 probability level.
to be 95.9 and 50.4 days at 25 and 40 °C, respectively. Immobilization of b-glucosidase on various soil colloids enhanced the thermal stability at all temperatures (Table 3). In this case, the r2 correlation coefficients were higher than 0.98, with 95% confidence, despite some important deviation between the model prediction and the experimental half-lives. Results supported that the thermal stability of free b-glucosidase drastically decreased with temperature and can be rapidly denatured. Coarse soil colloid-immobilized b-glucosidases presented the higher thermal stability than fine colloids (Table 3). It probably meant that the thermal stability of the immobilized enzyme was greatly affected by the affinity between the b-glucosidase molecules and the surface of soil colloidal particles, the higher affinity with the higher thermal stability. This finding may be useful in guiding and designing enzyme immobilization engineering, and helpful for further research on enzymatic mechanisms in carbon cycling and soil carbon storage. Usually, the soil colloidal support has a protecting effect from heat when the immobilized enzyme inactivation occurs. The immobilization step affected the conformational flexibility and caused an increase in enzyme rigidity, which was commonly reflected by an increase in stability toward denaturation by raising the temperature [39]. 3.5. Effect of pH on activities of free and immobilized b-glucosidase Fig. 5. Semilogarithmic plots of remaining free and immobilized enzyme as a function of time of incubation at 25 °C (a) and 40 °C (b). The error bar at each data point represents the standard error (n = 3).
A similar pH–activity profile was observed for the free and immobilized enzyme in the range of pH 4.5–7.0 in Fig. 6a. Both free
Fig. 6. Effects of pH (a) and temperature (b) on activities of free and immobilized b-glucosidase. The error bar at each data point represents the standard error (n = 3).
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and immobilized b-glucosidase displayed the highest activity at pH 6.0. No obvious shift for the optimal pH of immobilized enzyme was observed as described previously by some investigators [38]. However, the decline of activity as pH increased from 6.0 to 7.0 or decreased from 6.0 to 4.0 was much more sharp for free b-glucosidase than immobilized b-glucosidase. Instead, more flat curves were observed for b-glucosidase immobilized on soil colloidal particles. These results suggested that free enzyme was more sensitive to pH changes than soil colloidal particle-immobilized enzymes. 3.6. Effect of temperature on activities of free and immobilized b-glucosidase The role of the immobilization on the catalytic behavior of b-glucosidase was evident by temperature–activity curves illustrated in Fig. 6b at temperatures from 17 to 77 °C. The figure clearly shows that both free and immobilized enzymes had an optimal activity at 57 °C. However, a smooth behavior (i.e., less dependence on temperature) was in general exhibited by b-glucosidase immobilized on soil colloidal particles, which indicated that the thermal stability of the immobilized b-glucosidase was higher than the free enzyme. As temperature increased from 57 to 77 °C, the activity of free b-glucosidase decreased by 31.2%; immobilized enzymes declined by 8.2–15.3%. Results suggested that the thermal stability of immobilized b-glucosidase becomes significantly higher than that of free b-glucosidase at higher temperatures. Immobilization of b-glucosidase on various soil colloids from a paddy soil is supposed to preserve the tertiary structure enzyme, and it protected the enzyme from conformational changes causing effects of the environment. The results also revealed that temperature–activity profiles of coarse colloid-immobilized enzymes were more flat than fine clays. For the same particle size of clay fractions, the thermal stability for organic clays was higher than that for inorganic clays. Similar results were reported by Rao et al. [40]. 4. Summary Higher adsorption amounts and lower adsorption affinity of b-glucosidase were found on fine soil colloidal particles, which were attributed to their higher surface area and higher content of iron oxides. b-Glucosidase adsorbed on coarse soil colloidal particles was also more easily released. More enzyme molecules were adsorbed on organic soil colloidal particles, confirming the facilitated effect of organic substances in the adsorption of enzymes. Decrease in the specific activity of b-glucosidase on immobilization indicated that some of the enzyme active sites were hindered by the soil colloidal particle surfaces, probably as the result of the inaccessibility or modifications of the active site of the enzymes. Immobilized b-glucosidase on soil colloidal particles was more stable and less sensitive to the aging process than the free b-glucosidase. In terms of their half-life, immobilization of b-glucosidase on various soil colloids enhanced the thermal stability at all temperature. Due to the protective effect of supports, soil colloidal particleimmobilized enzymes were also less sensitive to pH and temperature changes than free enzyme. Data obtained in this study are helpful for further research on enzymatic mechanisms in carbon cycling and soil carbon storage.
Acknowledgments This study was supported by Major Program of National Natural Science Foundation of China (No. 40830528) and the Natural Science Foundation of Department of Education, Jiangsu Province (08KJB210002). References [1] C. Trasar-Cepeda, F. Gil-Sotres, M.C. Leirós, Soil Biol. Biochem. 39 (2007) 311. [2] R.P. Dick, in: J.W. Doran, D.C. Coleman, D.F. Bezdicek, B.A. Stewart (Eds.), Defining Soil Quality for a Substainable Environment, Soil Science Society of America, Madison, WI, 1994. [3] C. Trasar-Cepeda, M.C. Leirós, F. Gil-Sotres, Soil Biol. Biochem. 40 (2008) 2146. [4] P. Nannipieri, E. Kandeler, P. Ruggiero, in: R.G. Burns, R.P. Dick (Eds.), Enzymes in the Environment, Dekker, New York, 2002. [5] R.G. Burns, Soil Biol. Biochem. 14 (1982) 423. [6] M.-C. Marx, E. Kandeler, M. Wood, N. Wermbter, S.C. Jarvis, Soil Biol. Biochem. 37 (2005) 35. [7] J.M. Sarkar, A. Leonowicz, J.-M. Bollag, Soil Biol. Biochem. 21 (1989) 223. [8] P. Nannipieri, L. Gianfreda, in: P.M. Huang, N. Senesi, J. Buffle (Eds.), Environmental Particles Structure and Surface Reactions of Soil Particles, Wiley, New York, 1998. [9] P. Nannipieri, P. Sequi, P. Fusi, in: A. Piccolo (Ed.), Humic Substances in Terrestrial Ecosystems, Elsevier, Amsterdam, 1996. [10] R.G. Burns, in: P.M. Huang, M. Schnitzer (Eds.), Interactions of Soil Minerals with Natural Organics and Microbes, Soil Science Society of America, Madison, WI, 1986. [11] M.D. Busto, M. Perez-Mateos, Eur. J. Soil Sci. 51 (2000) 193. [12] H. Quiquampoix, Biochimie 69 (1987) 753. [13] A.A. Safari-Sinegani, G. Emtiazi, H. Shariatmadari, J. Colloid Interface Sci. 290 (2005) 39. [14] S. Kiss, M. Draga-Bularda, D. Radulescu, Adv. Agron. 27 (1975) 25. [15] J. Skujins, CRC Crit. Rev. Microbiol. 4 (1976) 383. [16] J.N. Ladd, R.C. Foster, P. Nannipieri, J.M. Oades, in: G. Stotzky, J. Bollag (Eds.), Soil Biochemistry, vol. 9, Dekker, New York, 1996. [17] S.P. Deng, M.A. Tabatabai, Biol. Fertil. Soils 22 (1996) 208. [18] H. Quiquampoix, Biochimie 69 (1987) 765. [19] G. Geiger, H. Brandl, G. Furrer, R. Schulin, Soil Biol. Biochem. 30 (1998) 1537. [20] M.-Y. Chang, R.-S. Juang, Biochem. Eng. J. 35 (2007) 93. [21] IUSS Working Group WRB, World Soil Resources Reports No. 103, FAO, Rome, 2006. [22] Soil Survey Staff, Soil Survey Investigations Report No. 45, Version 1.0, USDANRCS, National Soil Survey Center, Lincoln, NE, 1995. [23] Q. Huang, W. Liang, P. Cai, Colloids Surf. B 45 (2005) 209. [24] Y. Xiong, Soil Colloids, vol. 2, Science Press, Beijing, 1983 (in Chinese). [25] M.A. Tabatabai, in: R.W. Weaver, J.S. Angel, P.S. Bottomley (Eds.), Methods of Soil Analysis, Part 2, Microbiological and Biochemical Properties, Soil Science Society of America, Madison, WI, 1994. [26] C.H. Giles, T.H. MacEwan, S.N. Nakhwa, D. Smith, J. Chem. Soc. 56 (1960) 3973. [27] S.A. Boyd, M.M. Mortland, in: J.-M. Bollag, G. Stotzky (Eds.), Soil Biochemistry, Dekker, 1990. [28] P. Ruggiero, J. Dec, J.-M. Bollag, in: G. Stotzky, J.-M. Bollag (Eds.), Soil Biochemistry, vol. 9, Dekker, New York, 1996. [29] S. Staunton, H. Quiquampoix, J. Colloid Interface Sci. 166 (1994) 89. [30] G. Geiger, M.P. Livinston, F. Funk, R. Schulin, Eur. J. Soil Sci. 49 (1998) 17. [31] P.G. Grossi, D.L. Sparks, C.C. Ainsworth, Environ. Sci. Technol. 28 (1994) 1422. [32] A.A. Safari Sinegani, G. Emtiazi, H. Shariatmadari, in: A. Faz, R. Ortiz, A.R. Mermut (Eds.), International Symposium on Sustainable Use and Management of Soils in Arid and Semiarid Regions, vol. II, Cartagena, Murcia, Spain, 2002. [33] A. Naidja, P.M. Huang, J.-M. Bollag, J. Mol. Catal. A: Chem. 115 (1997) 305. [34] G.V. Lubarsky, M.M. Browne, S.A. Mitchell, M.R. Davidson, R.H. Bradley, Colloids Surf. B 44 (2005) 56. [35] L. Gianfreda, M.A. Rao, A. Violante, Soil Sci. Soc. Am. J. 59 (1995) 805. [36] L. Gianfreda, M.A. Rao, A. Violante, Soil Biol. Biochem. 23 (1991) 581. [37] W.A. Dick, N.G. Juma, M.A. Tabatabai, Soil Sci. 136 (1983) 19. [38] S.K. Srivastava, K.S. Gopalkrishnan, K.B. Ramachandran, Enzyme Microbiol. Technol. 6 (1984) 508. [39] M.A. Abdel-Naby, Appl. Biochem. Biotechnol. 38 (1993) 69. [40] M.A. Rao, A. Violante, L. Gianfreda, Soil Biol. Biochem. 32 (2000) 1007.