Intensification in the Activity of Lipase Enzyme Using Ultrasonic

Jan 6, 2014 - Sonochemical Effect on Activity and Conformation of Commercial Lipases ... intensification in enzyme catalyzed reactions using ultrasoun...
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Intensification in the Activity of Lipase Enzyme Using Ultrasonic Irradiation and Stability Studies Sanket H. Jadhav and Parag R. Gogate* Chemical Engineering Department, Institute of Chemical Technology, Matunga, Mumbai 400 019, India S Supporting Information *

ABSTRACT: In the present work, the effect of ultrasound on the activity of lipase enzyme has been investigated using a probetype sonicator capable of delivering variable power at a constant frequency of 20 kHz. It was observed that lipase enzyme gave a maximum activity at an ultrasound intensity of 12.22 W/cm2 and an optimized sonication time of 9 min and any further increase in the intensity and time of sonication resulted in a decrease in activity. The maximum increase in the activity of the enzyme was 2-fold. All of the operating parameters were also optimized by the response surface methodology (RSM) approach. Immobilization of the enzyme was achieved after sonication at the optimized parameters to retain the activity of the enzyme, which gave 47.9% retention of the activity in the tributyrin hydrolysis reaction. Using the same reaction, the intensification phenomenon of the immobilized enzyme was also supported by thermodynamic studies, which showed considerable decreases in ΔG, ΔS, ΔH, and ΔE with sonicated enzyme as compared to the values obtained for reactions catalyzed with unsonicated immobilized enzyme. reactions.9 Ultrasound has been used in many biochemical applications such as cell disruption, microbial disinfection, crystallization, and emulsification of food materials.7,8 Fiametti et al.10 showed that the yields of monoacylglycerols and diacylglycerols increased with increasing power for a constant time of sonication and a constant enzyme concentration. It was observed that the use of ultrasound (40 kHz, 40 °C) promotes the passage of the protease through the pores of the leather skin and encourages the breakdown of the helical regions of collagen to accelerate the enzymatic hydrolysis by 25%.11 Wang et al.12 demonstrated that the activity of the enzyme allinase increased by 49% as a result of an increase in the intensity of ultrasound from 0.1 to 0.5 W/cm2. A further increase in the ultrasonic intensity to 0.7 W/cm2 however resulted in a decrease in the activity. Xiao et al.13 performed experiments to check the effect of varying frequency (40, 80, and 100 kHz) and power (100, 150, and 200 W) on the production of troxerutin esters. The results showed that the yield under continuous application of ultrasound at 80 kHz was 71.9% after 120 h, whereas the yields were 65.1% at 40 kHz, 61.2% at 100 kHz, and 57.4% under conventional shake flask conditions, confirming 80 kHz to be the optimum frequency. Barton et al.14 investigated the effect of ultrasound on sugar reducing enzymes and reported that, when an α-amylase starch solution was kept under application of ultrasound, a substantial increase in the rate of degradation was observed. Ozbek and Ulgen15 reported that the temperature increase due to sonication at 10% duty cycle was much lower than that at 90% duty cycle and the two modes gave different deactivation profiles for the enzyme. Li et al.16 also reported enhanced enzyme activities and rates of saccharification of the

1. INTRODUCTION Enzymatic reactions have been found to outperform chemical processes in terms of the specificity and performance feasibility of the processes. Currently, many industries such as the pulp, food, paper, and textile industries are using enzymes on a large scale for processing different products.1 Although enzymes are used to promote specific reactions, there are also some pitfalls in using enzymes, including higher costs; extended reaction times; and most importantly, the delicacy of enzymatic processes in terms of operation. However, some enzymes from mutated species or from species adapted to hot springs are stable up to 90 °C and hence can be directly used for reactions operated at higher temperatures to increase the reaction yields.2 Also, enzymes that are thermally stable can be easily pressure-stabilized and used for high-pressure reactions.3 Thermostable enzymes that can operate at high temperatures and give high yields can also be developed in research laboratories, but this process requires lots of physical and chemical modifications,4 as well as significant costs. Hence, there is a need for an alternative procedure to enhance the activity of normal enzymes. The stabilization of enzymes to increase their activity has been explored to a lesser extent. Certain approaches have been used to control the activity, including increasing the shear and temperature in the vicinity of the enzyme, and can be effectively used to intensify the rates of enzymatic reactions.5 There can be two ways to increase the yield of the product in any enzymatic process: One is to increase the transfer of the enzyme and substrate toward each other so that contact between them increases, and the other is to control the characteristics of the enzyme by altering its structure in response to a dynamic perturbation induced by some sudden change.6 Ultrasound, which is generally used for nondestructive testing, analytical purposes, and sonochemistry applications, can also be used for the intensification of enzyme activity and, hence, enzymatic © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1377

October 11, 2013 January 4, 2014 January 6, 2014 January 6, 2014 dx.doi.org/10.1021/ie403419e | Ind. Eng. Chem. Res. 2014, 53, 1377−1385

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pulp using cellulase enzyme in a stirred-tank reactor with continuous ultrasonic irradiation. Sulaiman et al.17 investigated the production of ethanol from lactose by fermentation with the yeast Kluyveromyces marxianus (ATCC 46537) under various sonication regimens and reported that the use of ultrasound under optimum conditions increased the yield by almost 3.5-fold. It was established that sonication at a controlled temperature can be used to substantially enhance the productivity of bioethanol fermentations. In another recent work, Sulaiman et al.18 reported that the rate of hydrolysis of soluble carboxymethyl cellulose in the presence of ultrasound under optimized conditions (sonication at 10% duty cycle and 11.8 W/cm2 power intensity) was nearly twice that of the control. Overall, it can be said that it is very important to optimize the parameters such as the power, frequency, time, and duty cycle of sonication to achieve the maximum activity of the enzyme. A brief overview of different studies related to the application of ultrasound on different enzymes and the effects of the main governing parameters are provided in Table S1 (Supporting Information).12−14,19−22 Although it is clear from the available literature that the activities of many enzymes increase in the presence of controlled sonication, to the best of our knowledge, stability studies of treated enzymes have not been reported at all in the open literature. It is expected that an enzyme tries to get back to its original conformation after the removal of influence of ultrasound and the activity of the treated enzyme can return to its normal state after some time. The main objectives of the present work include maximizing the intensification of enzyme activity by optimizing the ultrasound operating conditions and immobilization of the enzyme to avoid the loss of activity. Lipase was first treated by ultrasound under different conditions, which were optimized to maximize the intensification in the enzyme activity. Ultrasound treatment of a lipase preparation without the presence of a substrate enabled us to learn more thoroughly about the interaction between ultrasound and lipase enzyme. To explore the enzyme activation mechanism, the effects of ultrasound on the activity, kinetics, thermodynamics, and molecular structure of lipase were investigated with the aid of a chemical reaction kinetics model, the Arrhenius equation, Eyring transition state theory, and fluorescence spectroscopy. Such a detailed study into the effect of ultrasound on the lipase enzyme represents the novelty of the current work.

enzyme used was a standard pure enzyme, the experimental values are expressed in terms of enzyme activity. 2.2. Experimental Methodology. 2.2.1. Enzyme Assay. An acid−base titration assay was performed to determine the amount of butyric acid produced from the hydrolysis of tributyrin using lipase. The assay was performed in water because the enzyme is soluble in water. The pH of the assay mixture was 7.0, and the temperature for the assay was 35 °C. The method of assay is shown in Table 1. Continuous stirring is Table 1. Lipase Activity Assay Using Tributyrin as the Substrate step

reagent

1 phosphate buffer (50 mM, pH 7.0) 2 enzyme solution 3 tributyrin mix and stir continuously for 5 min on a cyclo mixer 4 add methanol indicator 5 alcoholic NaOH (0.05 M in methanol)

volume (μL) 1062 250 188 5000 a

a

Until the end point, indicated by a change from colorless to faint pink.

required to mix the two immiscible phases of triglyceride and water, and hence, the reaction mixture was stirred on a vortex mixer (model SA8) supplied by Stuart Equipment (Staffordshire, U.K.). The time and speed of rotation for mixing were kept the same for all samples to avoid inconsistencies due to mixing. Because the enzyme acts on the interphase, it is beneficial to use a well-mixed emulsion for a more efficient reaction. The reaction was stopped by deactivating the enzyme through the addition of methanol, along with phenolphthalein as an indicator. The mixture was titrated with 0.05 M alcoholic NaOH using a micropipet. 2.2.2. Sonication of Enzyme. The intensification of enzyme activity can be achieved by changing the environment surrounding the enzyme. As reported in the literature,23 enzymes are stabilized under high-pressure and high-temperature conditions. Ultrasonication induces higher local temperatures and local pressures in the medium which can be helpful to keep the enzyme stable under ultrasonic conditions. The ultrasonic device used for sonication was a probe sonicator. The ultrasonic irradiation at a frequency of 20 kHz was transferred through a titanium cylindrical horn. During operation, the bulk temperature of the system was maintained constant using a constant-temperature water bath, which was essential to nullify the possible interference of changes in temperature in altering the enzyme activity. 2.2.3. Measurement of Intrinsic Fluorescence. Two samples were prepared to measure intrinsic fluorescence. Sample 1 was free lipase enzyme without ultrasonic treatment. Sample 2 was lipase enzyme treated under optimized conditions of a power of 60 W for an irradiation time of 9 min . 2.2.4. One-Variable-at-a-Time Approach. Initially, the operating variables for the ultrasonic irradiations were optimized using the one-variable-at-a-time (OVAT) methodology. The advantage of this approach is to determine the effects of individual operating variables on the output variable without interference from the other variables. The temperature of the solution was kept constant at 28 °C using a water bath to avoid its effect on the reaction. Optimization of Ultrasound Intensity. Sonication was performed in direct mode with 100 mL of 3 mg/mL enzyme

2. MATERIALS AND METHODS 2.1. Materials. Standard lipase enzyme CALB was obtained from Novozymes, Bagsvaerd, Denmark. The activity of the enzyme was 152 HU/mL·min (where HU represents hydrolyzing units). Tributyrin, sodium dihydrogen phosphate, and disodium hydrogen phosphate were obtained from Himedia Laboratories, Mumbai, India. An ultrasonic horn having a supplied power rating of 220 W and an operating frequency of 20 kHz was obtained from Dakshin, Mumbai, India. Calorimetric studies, based on the method of monitoring the rise in the temperature of water with reference to the time of irradiation, were undertaken to quantify the actual power dissipation into the system. Based on the calorimetric study, the energy efficiency, which is described as the ratio of the actual power dissipated into the system to the total power supplied to the system (expressed as a percentage), was found to be 14%. All of the chemicals used were of analytical grade and were used as received from the supplier, unless otherwise specified. As the 1378

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To investigate this aspect, experiments were performed in which the sonicated enzyme solution was kept at room temperature for 1 h. Sampling was done every 5 min, and an enzyme activity assay was performed to check the change in activity with time under normal conditions. 2.2.7. Immobilization. Immobilization was done to ensure retention of the conformation of the enzyme obtained after sonication. Two different methods were used for immobilization to investigate the effects of different immobilization techniques on both the retention of activity and the overall stability of the enzyme after sonication. In the first approach, the unsonicated enzyme was entrapped in calcium alginate beads, whereas in the second method, the enzyme was physically adsorbed on a hydrophobic resin called HP20. As the latter method was found to be more effective, pH and adsorption studies were performed in detail for HP20 adsorption to achieve the maximum binding capacity. 2.2.8. Thermodynamic Studies. Thermodynamic parameters were obtained by performing experiments on the hydrolysis of tributyrin at varying temperatures to investigate the effect of temperature on the reaction yield. Sampling was done after every 10 min. The Arrhenius equation and Eyring’s transition state theory equation were used to obtain the thermodynamic parameters. Experiments were performed using both unsonicated immobilized enzyme (USIE) and sonicated immobilized enzyme (SIE) with the objective of comparing the thermodynamic parameters.

solution using an ultrasonic horn. The ultrasonic horn had a fixed operating frequency of 20 kHz. The horn was dipped in the solution to a 0.5 cm height. The ultrasound intensity was varied over the range from 6.11 to 16.30 W/cm2. The other parameters such as duty cycle and enzyme concentration were kept constant and away from their extreme values (based on the predefined range of parameters), so that the variation in activity was an exclusive function of power alone. Samples were withdrawn at regular time intervals and monitored for enzyme activity with the objective of optimizing the time of sonication in the same experiment for each power input. Optimization of Duty Cycle. It is very important to check the effect of application of intermittent ultrasound on the variation in the activity of the enzyme. Experiments were designed to check the effect of the duty cycle at the optimized ultrasound intensity. The concentration of enzyme was kept constant at 3 mg/mL, and samples were taken at different time intervals for analysis. The on and off times for the ultrasonic irradiation are listed in Table 2. The off time of the horn was kept constant at 5 s to reduce the risk of wear and tear on the horn. Table 2. Time Ratios for Varying Duty Cycle on time (s)

off time (s)

duty cycle (%)

2 4 10 20 45

5 5 5 5 5

28.50 44.50 66.67 80 90

3. RESULTS AND DISCUSSION The basic effect produced by ultrasound is to increase the accessibility of the substrate toward the enzyme. The microstreaming occurring during ultrasonication facilitates mass transfer, ensuring substrate availability. Ultrasound also alters the enzyme configuration to enhance its activity.24 3.1. Effects of Operating Variables. The effects of different operating variables on the increase in activity of the enzyme are discussed in the following subsections. 3.1.1. Effects of Ultrasound Intensity and Time of Sonication. Figure 1 shows the variation in the activity of the enzyme at different ultrasound intensities. It was observed that the activity of the enzyme increased continuously until an optimum intensity of 12.22 W/cm2 and a further increase in intensity led to a decrease in the activity of the enzyme. At an ultrasound intensity of 16.30 W/cm2, a total loss of activity of

Optimization of Enzyme Concentration. The reaction rate is generally proportional to the enzyme concentration, and hence, it increases with increasing enzyme concentration. Experiments with varying concentrations of enzyme in the range of 1.0−7.0 mg/mL were performed at the optimized ultrasound intensity and duty cycle to investigate the effect of concentration on the activity of enzyme in the presence of ultrasound. The amount of substrate taken was 188 μL. 2.2.5. Response Surface Methodology (RSM). All parameters were optimized using the one-variable-at-a-time (OVAT) approach. OVAT does not consider the interference of different parameters with each other, and hence, there is a need for a complete approach to optimize the conditions for intensification. Response surface methodology was used to create a surface of reaction output coordinates (i.e., activity in terms of hydrolyzing units) at different operating conditions. The exact ranges of the four operating variables were determined based on the OVAT analysis, and a spectrum of different experiments was created using DesignExpert 8.0 software. Thirty experiments were performed, and the results were analyzed and optimized using the software to create an optimized response surface. Table S2 (Supporting Information) lists the coded and uncoded variables for the experiments. Table S3 (Supporting Information) reports the number of experiments performed and the results obtained. 2.2.6. Stability Studies. It is very important to check the degree of intensification defined in terms of the increase in activity immediately after sonication. It is also important to check whether the enzyme retains the same activity when it is removed from the influence of ultrasound because it is possible that enzyme could return to the same conformation after removal of the environment applied at the time of sonication.

Figure 1. Effects of ultrasound intensity on enzyme activity (duty cycle, 66%; enzyme concentration, 3.0 mg/mL). 1379

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the enzyme was observed. Sulaiman et al.17 reported similar results for the production of ethanol from lactose by fermentation with the yeast Kluyveromyces marxianus (ATCC 46537) in the presence of ultrasonic irradiation. They reported that sonication at 10% and 20% duty cycles appeared to stimulate yeast growth compared to the control fermentation whereas a 40% duty cycle had a measurable adverse impact on cell growth. Cavitation produces free hydroxyl and hydrogen radicals by virtue of the pyrolysis of water. The observed behavior can be attributed to the reaction of hydroxyl or hydrogen radicals formed during ultrasonication with the protein backbone, which is a probable reason for protein denaturation.25 This phenomenon can subsequently lead to enzyme aggregation, thus obstructing the active sites and at the same time decreasing protein stability.26 Cavitation also forms hot spots,27,28 which create local conditions of high temperature (5000 K) and high pressure (>100 bar) that are detrimental to protein structure and function. Shear forces that occur in solution during ultrasound irradiation also have a significant role in enzyme inactivation.15 As can be seen in Figure 1, enzyme activity decreased after 9 min of ultrasound treatment, so 9 min was considered to be the optimum time of treatment at 12.22 W/ cm2 intensity. 3.1.2. Effects of Duty Cycle. Along with the time of sonication, the ultrasonic duty cycle is one of the main operating parameters. The results obtained for the effects of duty cycle on the activity of the enzyme are shown in Figure 2.

and effective operation, the duty cycle should be optimized as per the methodology described in the present work. 3.1.3. Effects of Enzyme Concentration. Enzyme concentration plays an important role in determining the effects of sonication. As shown in Figure 3, almost the same effect was

Figure 3. Effects of enzyme concentration on enzyme activity (power, 60 W; duty cycle, 66%).

observed for enzyme concentrations of both 4 and 5 mg/mL, but for increased concentrations beyond 5 mg/mL, the extent of intensification decreased. Basically, at very low concentrations, the enzyme molecules are so randomly distributed with very low density that they cannot effectively interact with microstreams generated due to acoustic cavitation continuously. Beyond the optimum concentration of the enzyme, excess enzyme molecules hinder the energy-transfer process, thereby decreasing the available energy for cavitational events.29 There is also a possibility of forming aggregates of enzymes as a result of cavitation, which can further lead to a lower degree of intensification. Even the enzyme kinetics also reveals similar effects that is, at higher concentrations of enzyme, there is competition for the substrate, which decreases the rate of reaction. 3.2. Response Surface Methodology (RSM). The results of RSM analysis were similar to the profiles obtained by OVAT analysis. The values obtained after all 30 experiments were optimized using the software for the highest response, namely, enzyme activity. Analysis of variance (ANOVA) suggested that the model was significant, with an R2 value of 0.951, and can mimic real experiments and predict the output of experiments with significant accuracy. It is important to note that the results obtained were significant in terms of the model statistical parameters. A model F value of 17.86 implies that the model is significant, as there is only a 0.01% chance that a model F value this large could occur due to the noise. Values of Prob > F that are less than 0.0500 indicate that all of the model terms are significant. In this case, the terms involving A, D, BD, A2, B2, and D2 were found to be the significant model terms. The model equation fitted in the present work is given by

Figure 2. Effects of duty cycle on enzyme activity (intensity, 12.22 W/ cm2; enzyme concentration, 3.0 mg/mL).

It can be seen from Figure 2 that the effects of duty cycle also depend on the time of sonication. At 28.5% duty cycle, the activity reached a maximum at 20 min of sonication, whereas at 66.67% duty cycle, the same maximum was reached in a shorter treatment time. Similar trends were also observed at 80% duty cycle. As less energy is required to maintain 66.67% than 80% duty cycle, 66.67% was chosen as optimized value. The reason for obtaining better results at 66.67% might be the appropriate application of impulsive forces on the enzyme. Below 66.67% duty cycle, it is difficult to achieve the effect of microstreaming in an effective manner. Duty cycle signifies how intermittently the enzyme is sonicated. Continuous sonication should ideally lead to faster intensification, but continuous sonication also generates more heat, which can lead to the degradation of the enzyme. The maintenance of the equipment is another concern. To maintain a balance between energy consumption

hydrolyzing units = 296.13 − 31.11A + 1.22B + 8.29C + 28.66D − 1.84AB − 8.35AC + 3.87AD + 4.28BC − 12.02BD + 1.84CD − 42.83A2 − 22.45B2 − 6.15C 2 − 24.49D2

where the variables A, B, C, and D correspond to power, time, duty cycle, and enzyme concentration, respectively. The obtained results are in accordance with the OVAT analysis, 1380

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Table 3. Validation of RSM Optimization enzyme activity (HU/mL·min) 1 2 3

power (W)

time (min)

duty cycle (%)

conc of enzyme (mg/mL)

predicted

experimental

desirability

deviation (%)

55.47 55.48 55.58

10.91 10.79 11.07

80.00 79.98 80.00

5.39 5.40 5.41

315.81 315.8 315.78

324 326 318

0.955 0.955 0.954

2.59 3.22 0.70

consist of mechanical, thermal, chemical, electrical, and acoustic methods.31 The important point to be noted here is that the activity of enzyme increased to 2.1 times the original value and then decreased to 1.24 times after 1 h and remained constant beyond this time. Thus, the use of ultrasound certainly provided some permanent intensification in the activity of the enzyme, as the activity of enzyme did not return to its original value, which also indicates that the reconfiguration of the enzyme might not follow the same path that it followed while undergoing change during sonication. It can also be said that the emulsion formed under ultrasound was much more stable for at least 2−3 h; hence, most of the contributing effects of ultrasound can be attributed to the change in the structure of the enzyme. 3.4. Effects of Ultrasonic Treatment on the Structure of Lipase. The maximum wavelength of the emission, λmax, of an enzyme depends on its microenvironment. The disruption of the native structure of lipase leads to changes in the exposure of tryptophan and tyrosine side chains to the solvent, which can be readily monitored by the protein fluorescence emission spectrum.32 In the present study, changes in the enzyme conformation caused by the application of ultrasound were investigated by tryptophan fluorescence (the maximum fluorescence emission wavelength is 345 nm), and the obtained results are presented in Figure 6. As can be seen in Figure 6, the

and the validation of the optimized data also showed very little deviation with the results predicted by the model. The obtained optimum parameters are reportesd in the Table 3 with the validation data. The surface created for variation of the hydrolyzing units with respect to the power and time of ultrasonic irradiation is shown in Figure 4.

Figure 4. Response surface for the variation of hydrolyzing units with power and time.

3.3. Stability of Enzyme after Sonication. The results obtained in the stability studies are shown in Figure 5. It can be

Figure 5. Changes in the enhanced enzyme activity after sonication.

Figure 6. Fluorescence spectra of untreated and ultrasound-irradiated lipase enzyme.

seen from this figure that, when a sonicated enzyme was kept at room temperature for some time, a steady and gradual decrease in the activity of the enzyme was observed. The decrease in the intensified activity can be attributed to two main causes: the reconfiguration of the enzyme toward its original state and a decrease in the surface area of the substrate because of deemulsification, which can decrease the extent of reaction of the enzyme. De-emulsification results in the separation of an emulsion into its component phases.30 De-emulsification is a two-step process involving flocculation followed by coalescence. Either of these steps can be rate-limiting in the overall breakage of the emulsion. Typical de-emulsification techniques

fluorescence intensity of the lipase enzyme decreased after ultrasonic irradiation as compared to that of the untreated enzyme, which clearly indicates that ultrasonic irradiation increased the number of tryptophan side chains on the lipase surface. However, the optimum fluorescence emission wavelength did not show a red or blue shift. These results indicate that the enzyme conformation changed with ultrasonic pretreatment. The explanation for this change could be that ultrasonic pretreatment induced conformational changes in the protein and destroyed the hydrophobic interactions of protein molecules, which, in turn, could cause more hydrophobic 1381

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binding. The constants of adsorption, including the maximum binding capacity (qmax) and coefficient of adsorption (K), were obtained from a graph of 1/q versus 1/C (where q is the amount adsorbed and C is the concentration) as presented in Figure 9. The qmax value for the resin was found to be 47.61 mg of enzyme/mL of resin, and the coefficient of adsorption was found to be 8.57 mg/mL.

groups and regions inside the molecules to be exposed to the outside.33 3.5. Immobilization Studies. Immobilization can be achieved through adsorption, entrapment, and covalent linkage. The immobilization process results in the stabilization of the enzyme in its current structure. As the stability of the enzyme after sonication in the changed conformation was very low, it was hypothesized that the stability could be retained by immobilization of the sonicated enzyme. With this background, studies were carried out to investigate the effect of immobilization on the activity of the enzyme. In the present work, immobilization of the enzyme by adsorption was studied in detail. 3.5.1. Optimization of pH for Physical Adsorption on HP20. Initially, the operating pH was optimized to achieve the maximum extent of adsorption, and the results obtained are shown in Figure 7. It can be seen that the maximum binding of

Figure 9. Fitting of the Langmuir adsorption isotherm for HP20.

Comparison of Immobilization Approaches. The stability of an immobilized enzyme is determined by many factors such as the number of bonds formed between the enzyme and the carrier, the nature of the bonds (covalent, adsorption, crosslinking), and the conformation of binding. It was reported by Cao34 that the use of macroporous carriers keeps the enzyme more stable and active for reactions. The data obtained for the comparison of immobilization approaches are provided in Table 4. It can be seen that more binding occurred during a shorter time period for calcium alginate immobilization than for physical adsorption, which required about 5 times more time. However, calcium alginate immobilization did not provide good activity when used in tributyrin hydrolysis, which can be attributed to the screening of the hydrophobic enzyme by hydrophilic residues, which does not allow much hydrophobic substrate such as oil or triglyceride to enter the hydrophobic active sites. Less availability of the substrate leads to decreased yields for the reaction. Physical adsorption showed better activity than entrapment by calcium alginate. In the case of physical adsorption using HP20, stretching of the enzyme takes place without interfering with the folding pattern of the enzyme. This stretching leads to stabilization of the enzyme on the surface of the matrix. Overall, physical adsorption leads to easy accessibility of the substrate to the enzyme active sites because of the macroporous nature of the resin and open, stretched structure of the enzyme. Hence, it was concluded that physical adsorption was a better approach in this case, so HP20 was used further for thermodynamic studies. 3.6. Thermodynamic Studies. It is well-known that the rate of reaction usually increases with an increase in the temperature of the reaction medium. This is because of the excess heat in the system, which is responsible for more random movement of the reactant particles, leading to an increase in interactions. The rate of reaction depends on the interactions between reacting molecules. In the case of enzymes, the availability of substrates to the enzyme is an important parameter for the reaction to occur. For the alcalase enzyme, it was shown by Ma et al.19 that the thermodynamic parameters and structural changes are interrelated. Alcalase enzyme under ultrasound undergoes transitions from tertiary to

Figure 7. Extents of adsorption of lipase at different pH values.

enzyme, which was 17.46 mg/mL of resin, occurred at pH 6. Considerable binding of 16.05 mg/mL of resin was observed at pH 7.0. The isoelectric point of the enzyme, where the enzyme is most hydrophobic, is in the range of 6.3−6.6. Ideally, maximum binding should be observed in this range of isoelectric pH values, but it was found that the enzyme precipitated and did not bind to the resin in this pH range. Based on the obtained results, the pH value used for further studies was 7.0, because all of the assays of the enzyme were acid−base titration assays carried out at pH 7.0. Adsorption Isotherm Studies. The obtained binding curve for HP20 resin is shown in Figure 8. It is clear from this figure that the Langmuir type of adsorption isotherm fitswell to this

Figure 8. Fitting of the adsorption isotherm for adsorption on HP20. 1382

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Table 4. Immobilization of Standard Lipase Enzyme CALB: Retention of Activity

1 2 a

free enzyme activitya (HU/mL· min)

immobilized enzyme activitya (HU/g·min)

IF

technique

time (h)

extent of immobilization (%)

USE

SE

USE

SE

before immobilization

after immobilization

physical adsorption on HP20 calcium alginate entrapment

5 1

59.60 70

163

342

152 132

224 164

2.098

1.479 1.242

USE, unsonicated enzyme; SE, sonicated enzyme.

intensification obtained in the enzyme activity after immobilization. Applying the Arrhenius equation of state

secondary structures and secondary to primary structures to make the enzyme more open for substrates. These structural changes are accompanied by decreases in ΔG, ΔH, ΔS, and ΔE values. It is obvious that, if a certain enzyme has a more open structure, it can interact with more substrate molecules so that a higher reaction rate will be obtained as compared to the normal enzyme. Figures 10 and 11 show the reaction kinetics of hydrolysis by unsonicated immobilized enzyme (USIE) and sonicated

k = Ae(−Ea / RT )

(1)

we constructed plots of ln K versus 1/T for both USIE and SIE to determine the value of ΔE. Figure 12 presents the Arrhenius

Figure 12. Comparison of Arrhenius plots for USIE and SIE.

plots for USIE and SIE, as well as the ΔE values obtained for both enzymes. To determine the other thermodynamic parameters, Eyring plots of ln(K/T) versus 1/T were made, based on the governing equation

Figure 10. Data for the hydrolysis kinetics at different temperatures for unsonicated enzyme.

⎛k T ⎞ ⎛ ΔS ⎞ ⎛ ΔH ⎞ ⎟ exp⎜ − ⎟ k = ⎜ B ⎟ exp⎜ ⎝ R ⎠ ⎝ RT ⎠ ⎝ h ⎠

(2)

Figure 13 shows a comparison of the Eyring plots for USIE and SIE. The Eyring plots resulted in good linear fits with ΔH extrapolated from the slopes of the curves and ΔS calculated from the intercepts. It was found that there was a change in the thermodynamic parameters of the enzyme after sonication.

Figure 11. Data for the hydrolysis kinetics at different temperatures for sonicated enzyme.

immobilized enzyme (SIE), respectively, at different temperatures. The positive difference between the rate constants of sonicated immobilized enzyme (SIE, 0.119) and unsonicated immobilized enzyme (USIE, 0.171) in the results obtained from the thermodynamic studies confirmed that there was definitely a change in the conformation of the enzyme that helped increase the rate of reaction. The ratio of rate constants was found to be 1.45, which is nearly equal to the

Figure 13. Comparison of Eyring plots for USIE and SIE. 1383

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variables, and experimental data sets used in RSM analysis. This material is available free of charge via the Internet at http:// pubs.acs.org.

Table 5 presents a comparison of all of the thermodynamic parameters for USIE and SIE. It can be seen that ΔG decreased

■

Table 5. Comparison of Thermodynamic Parameters parameter

unsonicated

sonicated

ΔE (J/mol) ΔH (J/mol) ΔS (J/mol·K) ΔG(318 K) (J/mol)

25332.76 22755.42 −192.12 5718.8

17318.06 14740.72 −215.05 5069.12

*Tel.: +91 22 33612024. Fax: +91 22 33611020. E-mail: pr. [email protected]. Notes

The authors declare no competing financial interest.

■

by 1.54% after ultrasonic treatment, which confirms the increase in lipase activity. The decrease in ΔH by nearly 8015 J/mol K can be attributed to the ultrasonically induced breakage of hydrogen bonds stabilizing the enzyme in the ground state and the disruption of the internal hydrophobic core, both of which accelerate the partial opening of the enzyme to expose more active sites. ΔS represents the difference in the extent of local disorder between the transition state and the ground state. The large decrease in the magnitude of ΔS can be attributed to the modification of amino acid residues and the initiation of cross-linking and aggregation leading to an increase in the enzyme activity. According to the second law of thermodynamics, any system tends to decrease the entropy to make reactions much easier. The entropy of the system decreased for the sonicated enzyme, and hence, it can be said that the sonicated enzyme will favor the reaction in much better way than the unsonicated enzyme. According to the obtained values, a considerable decrease in the ΔE value of the enzyme occurred after sonication, which supports the ease of reaction and, hence, should give better yields. This decrease in the activation energy confirms that changes occurred in the structure of the enzyme to expose active sites, favoring the easy access of the substrate to the enzyme.

REFERENCES

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4. CONCLUSIONS The important parameters for the intensification of lipase activity using sonication were optimized by the OVAT and RSM approaches. The obtained results indicate that, after sonication, the enzyme activity increased by 2-fold as compared to the original activity at an optimum intensity of 11.30 W/cm2, a total sonication time of 10.91 min, an 80% duty cycle, and an enzyme concentration of 5.39 mg/mL based on response surface methodology. The results also closely resemble the optimum parameters as obtained from the experimental investigations using the OVAT analysis. Immobilization was required to ensure the retention of activity, as well as the reuse of the enzyme. Immobilization was achieved by physical adsorption in a better manner than calcium alginate entrapment, and the enzyme exhibited a permanent increase in activity of ∼50% as compared to the original value. It was also found that the retention of the activity depends on the type of immobilization and the time required for immobilization. Thermodynamic studies indicated decreases in the values of ΔE, ΔS, ΔG, and ΔH, which shows that there was a definite change in the structural conformation of the enzyme as a result of sonication that led to a favorable change in the thermodynamic parameters to accelerate the reaction.

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