Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives

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JTICE-702; No. of Pages 10 Journal of the Taiwan Institute of Chemical Engineers xxx (2013) xxx–xxx

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Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives Ellappan Kalaiarasan, Thayumanavan Palvannan * Laboratory of Bioprocess and Engineering, Department of Biochemistry, Periyar University, Salem 636 011, Tamil Nadu, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 January 2013 Received in revised form 2 July 2013 Accepted 13 July 2013 Available online xxx

The bio-productive property of combinatorial polysaccharide additives (dextran and sodium alginate) on stability of horseradish peroxidase (HRP) for removal of phenols from acidic solutions was studied in this paper. The optimum pH range and temperature were determined for the stabilized enzyme as 3.6–5.4 and 65 8C, respectively. Enzyme stabilization experiments were conducted in the solution state without enzyme immobilization or encapsulation. The combinatorial polysaccharides were selected to construct an appropriate response surface methodology (RSM) for maximum HRP stabilization together with sodium acetate buffer to optimize the polysaccharide additives. The RSM results suggest 10.08% of dextran, 0.41% of sodium alginate and 64 mM sodium acetate buffer for maximum HRP stabilization at 65 8C with a predicted percentage residual activity of 60.01%. DSC results corroborated that the denaturation temperature (TD) values of stabilized HRP to be 30 8C higher than that of the native enzyme. The effect of pH on phenol removal for both native and stabilized HRP suggested that stabilized HRP exhibited high phenol removal activities even under acidic environment and successfully removed phenols. ß 2013 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Horseradish peroxidase Additives Stabilization Response surface methodology Differential scanning calorimetry Phenol removal

1. Introduction Aromatic compounds such as phenol, chlorophenol and its derivatives are one of the most common toxic pollutants and potent carcinogens. Widely varying levels of numerous aromatic compounds are present in the industrial effluents from petroleum refining, textiles, plastics, iron and steel [1]. Enzymatic removal of phenols has been reported by several investigations and it has been shown that peroxidases are able to react with aqueous phenolic compounds and form non-soluble materials that could be easily removed from the aqueous solution [2]. Among most abundant peroxidases, Horseradish peroxidase (HRP, EC 1.11.1.7) has been widely used in many studies for successful removal of phenolic compounds from aqueous medium, however; these detoxification processes suffer from enzyme inactivation [2,3]. Peroxidases tend to lose their stability when thermally induced at high temperatures [4,5] and inactivated within minutes under acidic conditions [6,7]. To overcome these types of problems, several strategies have been chosen such as immobilization, crystallization, stabilization, etc. [3,8]. Among them, the stabilization of soluble enzymes by

* Corresponding author. Tel.: +91 427 2345766/427 2345520; fax: +91 427 2345124. E-mail address: [email protected] (T. Palvannan).

using polysaccharide additives has been chosen in the literature [9]. Dextran is a flexible polysaccharide additive and its aldehyde derivatives have been used to cross link multimeric enzymes generating hydrophilic environments [10,11]. Sodium alginate is an exopolysaccharide produced mainly from brown seaweeds and it widely used as stabilizers, thickeners and gelling agents [12]. They are linear polymers of (1!4)-b-D-mannuronopyranosyl and (1!4)-a-L-guluronopyranosyl units in a copolymer that contains homopolymeric sequences [12–14]. The composition of monomers and their sequential character affects the gelating behaviour of sodium alginate [15,16]. The transition from water-soluble sodium alginate to water insoluble calcium alginate is based on its gelforming ability through cation binding, as the G-rich samples generally form hard and brittle gels in presence of Ca2+ [15,16]. Since Ca2+ induces the gelation behaviour of sodium alginate many investigations have reported on thermostability and phenol removal efficiency with immobilized HRP in calcium alginate, however; no study has been reported on thermostabilization and phenol removal efficiency of HRP from acidic solution without enzyme immobilization or encapsulation [2,3]. The use of polysaccharides as additives can avoid the destabilization of enzyme molecules against denaturation effects and also induces long shelf life [2,3,10]. These additives exert stabilizing effects by inducing protein hydration. Studies have been carried out to

1876-1070/$ – see front matter ß 2013 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jtice.2013.07.003

Please cite this article in press as: Kalaiarasan E, Palvannan T. Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives. J Taiwan Inst Chem Eng (2013), http://dx.doi.org/10.1016/ j.jtice.2013.07.003

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JTICE-702; No. of Pages 10 E. Kalaiarasan, T. Palvannan / Journal of the Taiwan Institute of Chemical Engineers xxx (2013) xxx–xxx

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RSM Zi zi

Dzi xi Y CCD DSC TD

response surface methodology uncoded value of ith independent variable uncoded value of ith independent variable at centre point step change value coded value of ith independent variable predicted response central composite design differential scanning calorimetry denaturation temperature

understand the interaction between the enzyme and polysaccharides, which results in the formation of complexes that improve enzyme stability in solution state [17]. The protein–polysaccharide interactions in aqueous solutions are susceptible to form either soluble or insoluble complexes depending on the electrical characteristics of the biopolymers and the solution composition [12]. In this article attempts have been made to optimize additives (dextran and sodium alginate) concentration for maximum stabilization of HRP for removal of phenols successfully from acidic environment. To achieve this object, HRP was stabilized and optimized by RSM. In this article, DSC measurements have been made to show the denaturation temperature of HRP in the presence and absence of additives. 2. Materials and methods 2.1. HRP assay The rate of decomposition of hydrogen peroxide (H2O2) by HRP, with guaiacol as hydrogen donor, was determined by measuring the rate of colour development colorimetrically at 436 nm and at 25 8C [18]. Lyophilized powder of purified HRP was purchased from Sigma. To obtain HRP the solid protein was dissolved in 100 mM of sodium acetate buffer at pH 4.2. The assay mixture (1.5 ml) contained 0.8 U/ml of HRP, 100 mM sodium acetate buffer (pH 4.2), 0.018 M of guaiacol and 10 mM of H2O2. Protein was determined by Lowry’s method [19] with bovine serum albumin as the standard. 2.2. Effect of pH and temperature on enzyme stability Stability assays were carried out in sealed tubes containing 0.8 U/ml of HRP in a total volume of 1.5 ml in 100 mM of sodium acetate buffer within a temperature controlled-thermo mixer comfort (eppendorf) at 45 8C. The combination of two polysaccharides (dextran, 12% and sodium alginate, 0.5%) with individual control was tested for a period of 24 h and the activity was measured periodically. To determine the optimum pH for native and stabilized HRP under acidic conditions, stability assays

were carried out at different pH values from 3.6 to 5.4. The optimum temperature for the stabilized enzyme was determined by measuring activity at different temperatures in the range of 30 to 70 8C at pH 4.2. All the chemicals used are commercially available and were used without purification. 2.3. Optimization of HRP stabilization by RSM 2.3.1. Central composite design (CCD) Response surface methodology (RSM) is a collection of statistical techniques for design of experiments that use quantitative data from appropriate experiments to determine regression model equations and operating conditions. The process variables under investigations are necessary to optimize the selected response [20,21]. A standard RSM design called CCD was applied in this investigation to find out the optimum effective variables for the maximum stabilization of HRP at 65 8C. The independent variables selected for this study was dextran (X1), sodium alginate (X2) and sodium acetate buffer (X3). Based on the results obtained in preliminary experiments dextran, sodium alginate and sodium acetate buffer were found to be the major variables which modulate the enzyme thermostabilization. Hence these variables were selected to find the optimized conditions for maximum thermostabilization of HRP using central composite design of RSM [22,23]. The DESIGN-EXPERT (Software) ver 6.0, was used for regression and graphical analysis of the data obtained. The centre points are used to determine the experimental error and the reproducibility of the data. The independent variables are coded to the (2, 2) interval where the low and high levels are coded as 2 and +2 respectively. The axial points are located at (a, 0, 0), (0, a, 0) and (0, 0, a) where a is the distance of the axial point from the centre and makes the design rotatable. In this study, the a value was fixed at 2 (rotatable). The range and the levels of the experimental variables investigated in this study are given in Table 1. For each categorical variable, a 23 full factorial central composite design (CCD) for the three variables, consisting of 8 factorial points, 6 axial points and 6 replicates at the centre points were employed leading to a total number of 20 experiments was used in this study. The number of experimental runs calculated from the following equation.

N ¼ 2n þ 2n þ x0 ¼ 23 þ 2  3 þ 6 ¼ 20 where N is the numbers of experiments required, n is the number of variables and x0 is the number of central points. To optimize the selected response, the optimum values of the selected variables were obtained by solving the regression equation and also by analyzing the response surface model. The effects of uncontrolled factors were minimized by randomized experimental sequence. The three variables were dextran (X1), sodium alginate (X2) and sodium acetate buffer (X3). The central values (zero level) chosen for experiment design were dextran (10%), sodium alginate (0.4%) and sodium acetate buffer (60 mM).

Table 1 Experimental range and levels of independent additive variables (e.g.: 3 factors). Z variables

Dextran (%) Sodium alginate (%) Sodium acetate buffer pH, 4.2 (mM)

Coded variables

X1 X2 X3

Variable levels 2

1

0

1

2

8 0.3 20

9 0.35 40

10 0.4 60

11 0.45 80

12 0.5 100

Step change value, DZi 1 0.05 20

Please cite this article in press as: Kalaiarasan E, Palvannan T. Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives. J Taiwan Inst Chem Eng (2013), http://dx.doi.org/10.1016/ j.jtice.2013.07.003

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JTICE-702; No. of Pages 10 E. Kalaiarasan, T. Palvannan / Journal of the Taiwan Institute of Chemical Engineers xxx (2013) xxx–xxx

Second degree polynomials, which include all interaction terms, were used to calculate the predicted response by the following equation. !2 n n n1 X n X X X bi xi þ bii xi þ bi j xi x j Y ¼ b0 þ i¼1

i¼1

i¼1 j¼iþ1

where Y is the predicted response, b0 the constant coefficient, bi the linear coefficients, bij the interaction coefficients, bii the quadratic coefficients and xi, xj are the coded values of HRP stabilization variables [23]. Statistical analysis of the model was performed to evaluate the analysis of variance (ANOVA).

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dialyzed using a 64 mM sodium acetate buffer (pH 4.2) at 4 8C, by three buffer changes for 3 h [28]. The thermal treatment of HRP was carried out at 65 8C for a period of 24 h in sodium acetate buffer (64 mM, pH 4.2). Partially purified extract of HRP enzyme was used as a control. The thermostability of HRP was visualized by incubating the gel for 3 min at room temperature in sodium acetate buffer (64 mM, pH 4.2) in the presence of guaiacol (0.018 M) and 10 mM of H2O2 followed by staining with coomassie blue to determine the stability of HRP and their thermal inactivation pattern. 2.7. Optimization of parameters for phenol removal using stabilized HRP

2.4. Storage and thermal stability assay The additive effect of combined polysaccharides, dextran (10.08%) and sodium alginate (0.41%) on storage and thermal stability of HRP (0.8 U/ml) was investigated in sodium acetate buffer solution (64 mM, pH 4.2). For storage stability assays, we carried out the experiment in the solution state by incubating the HRP with optimized additives at room temperature for 30 days and the residual activity of the HRP was determined by 10 mM of H2O2 and guaiacol (0.018 M). The controls were run concurrently without any additives. The thermal stability of HRP in the presence of optimized and non-optimized additives were determined by the inactivation rate constant (k) as a function of temperature, between 30 and 65 8C and residual activity values. The temperature dependence of k was analyzed from Arrhenius plot (natural logarithm of k versus reciprocal of the absolute temperature); the activation energy (Ea) was obtained from the slope of the plot [24]. This is a constant and does not vary with temperature. The slope of the line is equal to the negative activation energy divided by the gas constant, R (8.3145 J/mol/K) [24,25]. 2.5. Thermally induced conformational changes in HRP Differential scanning calorimetry (DSC) is one of the most frequently used techniques in studying the thermal stability of proteins. DSC investigates the thermal transitions in solution and to study the thermodynamics of protein folding [17,26,27]. Lyophilized powder of purified HRP was purchased from Sigma. To obtain HRP the solid protein was dissolved in 64 mM of sodium acetate buffer at pH 4.2 and the total protein was estimated by Lowry’s method [19]. The enzyme concentration used was 0.8 U/ml. The combined effect of optimized polysaccharide additives (dextran, 10.08% and sodium alginate, 0.41%) on HRP stability was investigated under solution state. DSC experiments were carried out to determine the denaturation temperature TD (8C) of each native and stabilized HRP in sodium acetate buffer (64 mM, pH 4.2). The buffer was used as a reference in all experiments. The denaturation temperature TD (8C) associated with the phase change for native HRP and stabilized HRP was measured under N2 atmosphere. The samples were heated from 10 to 120 8C at a scanning rate of 10 8C/min and the sample weight was 0.2 mg. The Shimadzu MicroCal DSC – 60 was used for the measurements. DSC data were analyzed using Origin version 4.0 software. 2.6. Non denaturing polyacrylamide gel (PAGE) 12% of Native PAGE was performed to determine the stability of HRP and the protein bands were stained with specific HRP staining and coomassie blue, respectively. Partially purified HRP was used in this study to determine the effect of additives (sodium alginate, 0.41% and dextran, 10.08%) on enzyme stability. HRP was extracted from horseradish roots (500 g) and crushed in a wet grinder with the addition of 64 mM sodium acetate buffer (pH 4.2) and the extract was centrifuged at 10,000 rpm for 6 min at 4 8C. The precipitate was

In order to determine the phenol removal efficiency using stabilized HRP, batch experiments were carried out by varying process parameters such as pH, enzyme concentration, phenol concentration and contact time. Briefly, purified HRP (Sigma) was dissolved in 64 mM of sodium acetate buffer at pH 4.2 and the total protein was estimated by Lowry’s method [19]. During the investigation, the assay was performed at 30 8C by adding 64 mM sodium acetate buffer, pH (4.2) containing 0.1 mM of phenol, 0.8 U/ml of enzyme and additives (dextran, 10.08% and sodium alginate, 0.41%). All the batch experiments were carried out in 25 ml conical flask containing 10 ml total reaction mixture. The controls were run concurrently for each set of treatments without any additives. In order to initiate the reaction, 1.5 mM H2O2 was added to the final reaction mixture and kept in the orbital shaker (Rivo Teck) at 120 rpm for 3 h. After 3 h 1 ml of sample was withdrawn and immediately mixed with concentrated dose of enzyme catalase (0.4 mg/ml) to stop the reaction and then we measured the concentration of phenol. The concentrations of phenol were determined by using a colorimetric assay in which the phenol within a sample reacts with 1 mM of 4-aminoantipyrene in the presence of potassium ferricyanide reagent, 3.5 mM. The absorbance of the assay mixture was measured at 510 nm after 10 min of incubation at room temperature [1,2]. Initially one parameter was evaluated and it was incorporated at its optimized level in the subsequent experiments. Initially, in order to determine the effect of pH on enzymatic degradation rates the experiment was carried out over the pH range 3.6–5.4 in a series of 25 ml conical flask containing 10 ml total reaction mixture (using 0.1 mM of phenol, 0.8 U/ml of HRP, additives (dextran, 10.08% and sodium alginate, 0.41%) and 1.5 mM H2O2) with constant agitation (120 rpm) for 3 h at 30 8C. In the same way effect of enzyme concentration was carried out by varying the enzyme concentration from 0.1 to 1 U/ml. The requisite HRP concentration was achieved by diluting the enzyme solution with 64 mM sodium acetate buffer, pH 4.2. In the same way subsequent experiments were carried out by varying the phenol concentration from 0.05 to 0.3 mM. Finally, experiments were conducted to know the contact time required for maximum phenol removal at optimized conditions of phenol (0.1 mM), enzyme concentration (0.9 U/ml) and pH 4.2 at 30 8C. Every 30 min, a 1 ml of sample was withdrawn from the flask, mixed with enzyme catalase (0.4 mg/ml) to stop the reaction and was assayed for phenol concentration (% removal) as described above. At the end of the test period, the sample mixtures were centrifuged at 4000 rpm for 30 min prior to measure the concentrations of the phenol. The supernatant was analyzed for residual phenolic compounds. 3. Results and discussion 3.1. Effect of pH and temperature on HRP stability HRP tends to lose their stability at 45 8C [10,11] and inactivated within minutes under acidic conditions [7,29]. The residual activity

Please cite this article in press as: Kalaiarasan E, Palvannan T. Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives. J Taiwan Inst Chem Eng (2013), http://dx.doi.org/10.1016/ j.jtice.2013.07.003

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JTICE-702; No. of Pages 10 E. Kalaiarasan, T. Palvannan / Journal of the Taiwan Institute of Chemical Engineers xxx (2013) xxx–xxx

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Fig. 1. Effect of pH on the stability of HRP activity in presence of individual (sodium alginate, 0.5% or dextran, 12%) and combined additives (dextran, 12% and sodium alginate, 0.5%). Experimental conditions were 45 8C for 24 h in 100 mM of sodium acetate buffer.

of HRP as function of the effect of pH in the presence and absence of additives (dextran, 12% and sodium alginate, 0.5%) are given in Fig. 1. After 24 h incubation in 100 mM of sodium acetate buffer at 45 8C, the enzyme was found to be more stable under acidic conditions with optimum stability at pH 4.2 in the presence of sodium alginate showing 91% of activity and with dextran the optimum stability was observed at pH 4.8 with 42% activity, respectively. The enhanced stability of the enzyme was observed in response to the combination of sodium alginate and dextran (97%) with optimum stability at pH 4.2 than with respect to the individual performance of sodium alginate and dextran, respectively. This appears to be surprising considering this was the average pH value for stabilized HRP. The temperature stability of HRP in the presence and absence of combinatorial additives was tested at 30, 40, 50, 60, 65 and 70 8C in sodium acetate buffer (100 mM, pH 4.2) for a period of 24 h (Fig. 2). The native HRP completely lost all its activity at 40 8C but relatively stabilized enzyme retains its activity up to 65 8C. The residual activity of stabilized enzyme was found to be about 98%, 97%, 96%, 86%, 42% and 5% at temperature values of 30, 40, 50, 60, 65 and 70 8C, respectively. These results showed that HRP has significant resistance to heat and acid pH in stabilizing form than it was in the native form. Regarding the role of polysaccharides on enzyme stability, previous studies have suggested that the interaction of proteins with polysaccharides to form complexes (soluble or insoluble complexes) may be stabilized predominantly by electrostatic, ion-dipole or hydrophobic interactions [12,29]. The thermostabilization by polysaccharide additives had been used to prevent the denaturation of enzyme at different pH and in fact variations in pH play an important role on enzyme stability [6,7,11,30]. Protein stabilization with polysaccharide additives is widely used strategy to form either soluble or insoluble complexes due to the electrostatic interaction between protein and polysaccharides [12,29]. Insoluble complexes were formed due to strong electrostatic attraction between the two biopolymers at

Fig. 2. Effect of temperature on the stability of HRP in the presence of combinatorial additives (dextran, 12% and sodium alginate, 0.5%). Experimental conditions were pH 4.2 and incubation for 24 h in 100 mM of sodium acetate buffer.

acidic pH when the protein and polysaccharide had the opposite electrical charges and no complexes were formed at basic pH when the protein and polysaccharide had similar electrical charges because of strong electrostatic repulsion between the two biopolymers [12]. Electrostatic forces drive interactions of charged biopolymers in aqueous solutions [12]. Analysis of HRP stability assays showed that dextran and sodium alginate as polysaccharide additives has the high stabilizing effect on HRP and we infer that these additives enhance the stability of enzyme at high temperatures under acidic environment. 3.2. Response surface methodology The results of the second order response surface model fitting as analysis of variable (ANOVA) are given in Table 2: The Model F-value of 14.46 infers that the model is significant. The Fisher F-test with a very low probability value, P = 0.0001 demonstrates a very high significance of the regression model. The Values of ‘‘Prob > F’’ less than 0.0500 indicate model terms are significant. The goodness of fit of the model was checked by the determinant coefficient (R2). The value of determinant coefficient R2 is 0.9287, indicates that only 8% are not explained by this model. The value of the adjusted determination coefficient (Adj. R2 = 0.8644) is also very high, confirms the significance of the model. ‘‘Adeq Precision’’ measures the signal to noise ratio. A ratio greater than 4 is desirable. Adeq precision ratio of 11.439 indicates an adequate signal. This model navigates the design space. Lower value of co-efficient of variation (C.V.% = 17.07) shows the improved accuracy and reliability of the conducted experiments [20–23]. Tables 3 and 4 depict the optimum predicted levels of dextran (%), sodium alginate (%) and sodium acetate buffer (mM)

Table 2 Analysis of variance (ANOVA) for selected model. Source

Sum of squares

Degree of freedom

Mean square

Model Residual Lack of fit Pure error Cor total

5130.27 394.17 394.08 0.092 5524.44

9 10 5 5 19

570.03 39.42 78.82 0.018

F value

P-value (Prob > F)

14.46

0.0001

4285.77

<0.0001

Predicted residual sum of squares (PRESS) = 3145.93, R2 = 0.9287, Adj. R2 = 0.8644, predicted R2 = 0.4305, Adeq precision = 11.439 and C.V.% = 17.07.

Please cite this article in press as: Kalaiarasan E, Palvannan T. Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives. J Taiwan Inst Chem Eng (2013), http://dx.doi.org/10.1016/ j.jtice.2013.07.003

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JTICE-702; No. of Pages 10 E. Kalaiarasan, T. Palvannan / Journal of the Taiwan Institute of Chemical Engineers xxx (2013) xxx–xxx Table 3 Levels of variables on HRP stabilization at 65 8C. Factors

Name

Level

Low level

High level

Std. Dev.

Coding

X1 X2 X3

Dextran Sodium alginate mM

0.077 0.23 0.20

1.00 1.00 1.00

1.00 1.00 1.00

0.000 0.000 0.000

Actual Actual Actual

The Y is response, the HRP stabilization expressed in logarithmic values, and X1, X2 and X3 are the coded values of the tested variables (dextran, sodium alginate and sodium acetate buffer) [20]. In developing the regression equation the independent variables were coded according to the equation: xi ¼

Table 4 HRP stabilization based on the variables. Response

Prediction

SE mean

95% CI low

95% CI high

SE Pred

95% PI low

95% PI high

R1

60.0168

2.53

54.37

65.66

6.77

44.93

75.10

for HRP stabilization at 65 8C for 24 h using RSM. Higher significance of the squared terms (X12 ; X22 and X32 ) followed by linear terms (X1, X2 and X3) and corresponding interaction terms (X1  X3, X2  X3, X1  X2) show that optimum values for stabilization of HRP lies within the experimental values chosen (Table 5). Insignificant interaction terms (X1  X3, X2  X3, X1  X2) can be neglected from the model without disturbing the integrity of the model. Two dimensional response surface plots (Fig. 3(a)–(c)) showed the optimum centre points for the maximum stabilization of HRP. Optimum interaction dextran (%) and sodium acetate buffer (mM) required for selected response (HRP stabilization) was shown in Fig. 3(a), where sodium alginate (%) was fixed at zero level. Fig. 3(b) reveals that existences of an optimum centre point for HRP stabilization which indicated that the good interaction of selected individual variables (i.e. sodium alginate (%) and sodium acetate buffer (mM)) with each other, where dextran (%) was fixed at zero level. Optimum interaction sodium alginate (%) and dextran (%) required for HRP stabilization, where sodium acetate buffer (mM) concentration was fixed at zero level Fig. 3(c). Fig. 4 shows the overlay plot which was constructed to show the effects of the interaction variables on the HRP stabilization. The overlay plot corroborated that there is an optimal point for the thermostabilization of HRP with selected variables. The maximum stabilization of HRP was achieved by selected variables and had a good interaction with each other. However, mild deviation of variables from optimal points for HRP stabilization altered the maximum stabilization of HRP. The application of response surface methodology follows regression equation which is an empirical relationship between the logarithmic values of HRP stabilization and tested variables in coded unit. Y ¼ 58:95 þ 3:21X 1 þ 5:95X 2 þ 3:19X 3  3:62X 1 X 2  1:00X 1 X 3  1:33X 2 X 3  11:54X12  10:47X22  10:47X32

(1)

5

zi  zi Dz i

(2)

where zi stands for the uncoded value of ith independent variable, zi denotes uncoded value of ith independent variable at centre point and Dzi is a step change value. Eq. (1) can be converted into the coded unit where, 0:08 ¼

zi  10 1

(3)

0:23 ¼

zi  0:4 0:05

(4)

0:20 ¼

zi  60 20

(5)

Response surface plots as a function of two factors at a time, maintaining all other factors at zero levels, are more helpful in understanding both the main and the interaction effect of these two factors [20,21]. Maximum and minimum principle of differential calculus was used to maximize Eq. (1) with respect to individual tested variables. The partial differential equations obtained are:

@Y ¼ 3:21  23:08X 1  3:62X 2  1:00X 3 @X 1

(6)

@Y ¼ 5:95  3:62X 1  20:94X 2  1:33X 3 @X 2

(7)

@Y ¼ 3:19  1:00X 1  1:33X 2  20:94X 3 @X 3

(8)

The second order differential equations are:

@2 Y ¼ 23:08 @X 1

(9)

@2 Y ¼ 20:94 @X 2

(10)

@2 Y ¼ 20:94 @X 3

(11)

The negative values of second order partial differential equations (Eqs. (9)–(11)) indicate the absence of local maximum and applicability of maximization. Eqs. (6)–(8) are equated to zero

Table 5 Significance of regression coefficients. Factor

Coefficient estimate

Degree of freedom

Standard error

95% CI low

95% CI high

Intercept X1 X2 X3 X1  X2 X1  X3 X2  X3 X12 X22 X32

58.95 3.21 5.95 3.19 1 1 1 1 1 1

1 1 1 1 2.22 2.22 2.22 1.65 1.65 1.65

2.56 1.70 1.70 1.70 8.57 5.95 6.27 15.23 14.15 14.15

53.24 0.57 2.17 0.60 1.32 3.94 3.62 7.86 6.78 6.78

64.65 7.00 9.74 6.97 1.00 1.00 1.00 1.02 1.02 1.02

X1, dextran; X2, sodium alginate; X3, mM.

Please cite this article in press as: Kalaiarasan E, Palvannan T. Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives. J Taiwan Inst Chem Eng (2013), http://dx.doi.org/10.1016/ j.jtice.2013.07.003

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Fig. 4. Overly plot depicting the interaction between sodium alginate and dextran for maximum stabilization of HRP. R1, residual activity (%). Actual Factor: X3, Sodium acetate buffer pH 4.2 (mM) = 0.20.

and solved for X1, X2 and X3, which give the maximum value of Y. 3:21  23:08X 1  3:62X 2  1:00X 3 ¼ 0

(12)

5:95  3:62X 1  20:94X 2  1:33X 3 ¼ 0

(13)

3:19  1:00X 1  1:33X 2  20:94X 3 ¼ 0

(14)

Algebraic solution to the above equations (Eqs. (12)–(14)) was X1 = 0.08, X2 = 0.23 and X3 = 0.20. These values correspond with the uncoded values of Z1 = 10.08%, Z2 = 0.41% and Z3 = 64 mM. In other words, 10.08% of dextran, 0.41% of sodium alginate and 64 mM of sodium acetate buffer has the collective effect on the thermostabilization up to 65 8C at pH 4.2. At these optimum values, the maximum predicted stabilization in terms of percentage of HRP stabilization was 60% at 65 8C. These optimum values are experimentally checked for validation and results were found to be 65% thermostabilization. The good correlation between these two results verifies the validity of the response model existence of an optimal point. 3.3. Storage and thermal stabilities of the free and stabilized HRP

Fig. 3. Two dimensional response surface plots show the optimum centre points for the maximum HRP stabilization. (a) Effect dextran and sodium acetate buffer pH, 4.2 (mM) on HRP stabilization is shown in the contour plot. (b) Effect of sodium alginate and sodium acetate buffer pH, 4.2 (mM) on HRP stabilization is shown in the contour plot. (c) Effect of sodium alginate and dextran on HRP stabilization is shown in the contour plot.

In the present study, we identified the optimum concentration of polysaccharide additives (dextran, 10.08% and sodium alginate, 0.41%) for maximum stabilization of HRP in sodium acetate buffer solution (64 mM, pH 4.2). This observation triggered us to identify the storage and thermal stability of HRP in the solution state itself. In general, enzymes are not stable during storage in solution and they tend to lose their activity gradually in acidic environment [6,7]. In order to determine the storage stability of HRP in acidic conditions we stored HRP in sodium acetate buffer solution (64 mM, pH 4.2) at room temperature for 30 days and the remaining activity of the enzyme was determined using guaiacol and H2O2. Fig. 5 shows the successful stabilization of HRP in the presence and absence of additives under the same storage

Please cite this article in press as: Kalaiarasan E, Palvannan T. Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives. J Taiwan Inst Chem Eng (2013), http://dx.doi.org/10.1016/ j.jtice.2013.07.003

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conditions. The free enzyme completely lost all its activity within 5 days and the stabilized HRP maintained about 76% of its initial activity during 30 days storage period. Thus, the stabilized HRP exhibits a higher stability than that of the free HRP. Because of this observation, thermal stability experiments were carried out in the presence of optimized and non-optimized additives, which were incubated without substrate at various temperatures. The activation energy required for the inactivation of HRP with optimized and non-optimized additives were calculated from the Arrhenius plot (Fig. 6) and it was found to be 17 and 14 kJ/mol, respectively. The significant differences in the slopes (activation energy) of Arrhenius plot in the presence of optimized and non optimized additives showed the differences in the mechanism of enzyme stabilization [24]. Significant change in the activation energy and the difference in the slope of Arrhenius plot in the presence of additives indicated that stabilization of HRP was of conformational origin and these studies will play a key role to understand the effect of temperatures of different enzymes. Thus these findings can be used to understand the innumerable application of HRP in diagnostics where it can function in multiple environments without storing at low temperatures because these stabilized enzymes can withstand long storage in acidic solution and at high temperature.

120

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3.4. Thermally induced conformation changes of HRP in the presence of additives In this paper, using DSC method the combinatorial effect of polysaccharide additives on thermostability of HRP were studied under acid pH, 4.2. The influences of pH on the thermal denaturation (TD) of enzymes were determined by using DSC analysis [29,31,32]. The typical DSC thermograms of HRP in the presence and absence of additives are shown in Fig. 7(a) and (b), respectively. During protein denaturation, the phase transition as changes in the lattice occurs with the absorption of heat [33]. The values of the denaturation temperature (TD) and the denaturation enthalpy (DH) for pure enzyme, in the absence and presence of additives, are tabulated in the Table 6. A TD of 52.71 8C was determined for native HRP without additives; addition of combinatorial additives (dextran, 10.08% and sodium alginate, 0.41%) yielded higher (83.56 8C) value of TD. These results agree well with the denaturation temperature measurements on pure HRP by differential scanning calorimetry (Fig. 7 and Table 6), which show that the enzyme is denatured at 52.71 8C and 83.56 8C in the absence and presence of additives, respectively. The DSC profiles depicts that a high amount of thermal stability of HRP was observed in the presence of polysaccharide additives. Regarding the role of polysaccharides on protein stabilization in an acidic environment, previous studies have reported in the literature that at acidic pH the negative charge on the polysaccharide was greater than basic, hence there are more binding sites are available for the positively charge proteins suggesting that the attraction between

Remaining activity (%)

100

80

60

Only HRP

40

Dextran + Sodium alginate stabilized HRP

20

0 5

10

15

20

25

30

Days Fig. 5. Storage stability of HRP in the presence and absence of optimized additives (dextran, 10.08% and sodium alginate, 0.41%) under room temperature for 30 days in sodium acetate buffer (64 mM, pH 4.2).

Fig. 6. Arrhenius plot for the effect of temperature on the activation rate constant of stabilized HRP with optimized and non-optimized additives, respectively in sodium acetate buffer (64 mM, pH 4.2).

Fig. 7. Thermal analysis effect of HRP in sodium acetate buffer (64 mM, pH 4.2) (a) without additives (b) with optimized additives (dextran, 10.08% and sodium alginate, 0.41%).

Please cite this article in press as: Kalaiarasan E, Palvannan T. Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives. J Taiwan Inst Chem Eng (2013), http://dx.doi.org/10.1016/ j.jtice.2013.07.003

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Table 6 Denaturation temperature (TD) and enthalpy (DH) for free and stabilized HRP in sodium acetate buffer (64 mM, pH 4.2). Factors

Denaturation temperature (TD)

DH, mJ (J/g)

Free HRP Stabilized HRP

52.71 8C 83.56 8C

6.08 mJ (0.40 J/g) 17.41 mJ (87.05 J/g)

two biopolymers strongly depends on their electrical characteristics and solution composition [12,29]. Differential scanning calorimetry has been used as a tool for rapid assay of the thermal stability of two Bacillus sp. a-amylases and horseradish peroxidase as a function of the concentration of glycerol, sorbitol and sucrose [34]. Thermal stabilization of glucose oxidase in the presence of water-soluble polymers has been also conducted with DSC as one of the useful technique to analyze the denaturation effect of temperature on enzymes. DSC has been widely used by several investigations to study the influence of pH associated with thermostabilization of HRP [29,35–37]. Therefore, the rise in denaturation temperature (TD) of HRP in the presence of polysaccharide additives reflects the enhanced stability of the enzyme under acidic conditions. The shifting of denaturation temperature (TD) demonstrates the endothermic reaction of the free and additives treated proteins which are an indicator of thermostability [34]. Proteins with higher melting point are less susceptible to unfold and denaturation at lower temperatures [38]. The transition enthalpy is actually a net value from a combination of endothermic contributions, such as the disruption of hydrogen bonds and exothermic ones, such as the break-up of hydrophobic interactions [38,39]. Analysis of our results provides evidence that polysaccharide additives play an important role in the thermostability of enzyme even under acidic environment. This is the first report signifying the ability of combinatorial additives (dextran and sodium alginate) to enhance the stability of HRP in the acidic solution itself. 3.5. Pattern of HRP stability The Native PAGE for thermal treatments of partially purified HRP in the presence and absence of combinatorial additives (dextran, 10.08% and sodium alginate, 0.41%) suggest higher stability and activity of stabilized HRP relative to the native one. The specific staining for HRP showed the presence of a band with partially purified enzyme in Fig. 8(a) suggests that HRP retains its activity after 24 h incubation with additives at 65 8C. Fig. 8(b) shows the Native PAGE pattern for thermal treatments of partially purified HRP with and without stabilizers with coomassie blue staining. For thermal treatments of partially purified HRP could explain the fact that combinatorial additives effectively stabilizes the enzyme in sodium acetate buffer (64 mM, pH 4.2) where the native HRP completely lost its activity. This study could explain the drastic change in activity of HRP and in fact this is the first report on the stability of HRP in solution state with combined additives (dextran and sodium alginate) without Ca2+ associations under acidic conditions. Studies are available on the analytical assessment of HRP activity through Native PAGE [40,41]. The present results were promising towards the application of combination of sodium alginate and dextran as a method for effective enzyme activity enhancement in an acidic environment. 3.6. HRP catalyzed removal of phenol In general, wastewater containing phenols and its derivatives exhibit high characteristic acidity and the removal of phenol from such solutions remains a challenge and hence the study has been conducted. The catalytic lifetime of a stabilized enzyme can be

Fig. 8. Native PAGE (12%) showing HRP stained with specific stain (a) and coomassie staining (b). (a) Partially purified HRP (lane 1), heat treated HRP at 65 8C for 24 h (lane 2) and HRP incubated in the presence of optimized polysaccharides at 65 8C for 24 h (lane 3). (b) Partially purified HRP (lane 1), heat treated HRP at 65 8C for 24 h (lane 2) and HRP incubated in the presence of optimized polysaccharides at 65 8C for 24 h (lane 3).

extended through an optimization of process variables that significantly affects enzyme activity. It was evident from the present work that the acid pH (3.6–5.4) significantly influenced the action of stabilized HRP in the presence of additives during phenol removal (Fig. 9(a)). The optimum pH value for phenol removal using stabilized HRP was observed at pH (4.2) with a maximum removal of about 51%. Since biocatalyst has a finite life time, normally phenol removal is dependent on the amount of catalyst added. To study the effect of enzyme concentration on phenol removal, different concentrations of HRP from 0.1 to 1 U/ml were used to compare the efficiency of stabilized enzyme. Fig. 9(b) shows the relationship between enzyme concentration and phenol removal. The removal efficiency of phenol increased with the increase in the concentration of HRP. It was found that for 0.1 mM of phenol concentration, increasing enzyme concentration from 0.1 to 0.9 U/ml results in the gradual increase in phenol removal. Further increase in the enzyme concentration has no significant effect on phenol removal. The enzyme concentration 0.9 U/ml was found to be the optimal dose for experiment condition and this concentration was fixed for the next set of experiments. Fig. 9(c) shows the influence of phenol concentration on HRP mediated phenol removal process in the presence and absence of additives. The removal rate was found to be increased up to certain concentrations of the phenol (0.1 mM). However, the phenol removal rate decreased above 0.1 mM of phenol concentration. These results depicts that when the amount of enzyme kept constant and the substrate concentration was gradually increased, the velocity of reaction increases until it reached maximum. After obtaining the equilibrium state any further addition of substrate did not alter the reaction rate. Furthermore, phenol removal experiments were carried out with stabilized enzyme to access the optimum contact time required for phenol removal. Studies were carried out at specific optimum reaction conditions of pH 4.2, 0.1 mM phenol concentration, 0.9 U/ml of HRP, additives (dextran, 10.08% and sodium alginate, 0.41%) and temperature 30 8C. At time intervals of 30 min each, the reaction mixture was analyzed for the phenol concentration (% removal) and data revealed that 150 min

Please cite this article in press as: Kalaiarasan E, Palvannan T. Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives. J Taiwan Inst Chem Eng (2013), http://dx.doi.org/10.1016/ j.jtice.2013.07.003

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Fig. 9. Influence of various process parameters on phenol removal efficiency of HRP with and without additives (a) pH (phenol concentration: 0.1 mM, additives: dextran (10.08%) and sodium alginate (0.41%), enzyme: 0.8 U/ml, agitation: 120 rpm, contact time: 3 h and temperature: 30 8C); (b) enzyme concentration (phenol concentration: 0.1 mM, additives: dextran (10.08%) and sodium alginate (0.41%), pH: 4.2, agitation: 120 rpm, contact time: 3 h and temperature: 30 8C); (c) phenol concentration (additives: dextran (10.08%) and sodium alginate (0.41%), enzyme: 0.9 U/ml, agitation: 120 rpm, contact time: 3 h, pH:4.2 and temperature: 30 8C); (d) contact time (phenol concentration: 0.1 mM, additives: dextran (10.08%) and sodium alginate (0.41%), enzyme: 0.9 U/ml, agitation: 120 rpm, pH:4.2 and temperature: 30 8C).

of reaction time is required to achieve maximum phenol removal (Fig. 9(d)). Studies on time course removal of phenol for both free and stabilized enzyme showed that stabilized enzyme successfully removes phenols from acidic solution when compared with the free enzyme. There are several studies reported on stabilization of HRP that include the addition of various polysaccharide additives [1,2,42,43], resulting in retention of HRP activity against physical parameters such as changes in pH and temperature. The use of additives can enhance phenol removal efficiency by forming a protective layer in the vicinity of the active centre of enzyme to restrict the attack of free phenoxy radicals formed in the catalytic cycle [44]. The calcium alginate immobilized HRP has a wide range of aromatic phenol removal in the industrial effluents and many studies has been conducted at various pH, as the phenol removal activity in waste water is highly pH dependent as it affects the degree of ionization [3,16]. We report for the first time the ability of combinatorial polysaccharide additives (dextran and sodium alginate) in stabilizing the HRP enzyme in solution state itself there by playing a critical role in phenol removal in acidic environment. 4. Conclusion In the present work, sodium alginate, dextran and sodium acetate buffer was successfully shown to stabilize HRP in an acidic environment against thermal degradation to study the activity of stabilized enzyme for removal of aqueous phenols. RSM which includes factorial design and regression analysis can better deal with multifactor influence on the experiments towards optimizing

the conditions for HRP stabilization. Results from thermal stability assays and DSC suggest that these optimized polysaccharides as additives exhibited increased stability of HRP up to 65 8C and at pH 4.2 in the solution state itself. We conclude the ability of stabilized HRP to retain a maximum optimal activity in acid pH for the removal of aqueous phenolic compounds in acidic solution which is the most common conditions in waste stream. Stabilized HRP was more stable during operation and storage when compared to native HRP. References [1] Liu JZ, Song HY, Weng LP, Ji LN. Increased thermostability and phenol removal efficiency by chemical modified horseradish peroxidase. J Mol Catal B Enzym 2002;18:225–32. [2] Alemzadeh I, Nejati S, Vossoughi M. Removal of phenols from wastewater with encapsulated horseradish peroxidase in calcium alginate. Eng Lett 2009;17:13. [3] Alemzadeh I, Nejati S. Phenols removal by immobilized horseradish peroxidase. J Hazard Mater 2009;166:1082–6. [4] Asada S, Torabib SF, Roudsaric MF, Ghaemia N, Khajehd K. Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase. Int J Biol Macromol 2011;48:566–70. [5] Hassani L, Ranjbar B, Khajeh K, Manesh HN, Manesh MN, Sadeghi M. Horseradish peroxidase thermostabilization: the combinatorial effects of the surface modification and the polyols. Enzyme Microb Technol 2006;38:118–25. [6] McEldoon PJ, Pokora RA, Dordick S. Lignin peroxidase-type activity of soybean peroxidase. Enzyme Microb Technol 1995;17:359–65. [7] Chattopadhyay K, Mazumdar S. Structural, conformational stability of horseradish peroxidase: effect of temperature and pH. Biochemistry 2000;39:263–70. [8] Qiu H, Lu L, Huang X, Zhang Z, Qu Y. Immobilization of horseradish peroxidase on nanoporous copper and its potential applications. Bioresour Technol 2010;101:9415–20. [9] Schmid RD. Stabilized soluble enzymes. Adv Biochem Eng Biotechnol 1979;12: 41–118.

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Please cite this article in press as: Kalaiarasan E, Palvannan T. Removal of phenols from acidic environment by horseradish peroxidase (HRP): Aqueous thermostabilization of HRP by polysaccharide additives. J Taiwan Inst Chem Eng (2013), http://dx.doi.org/10.1016/ j.jtice.2013.07.003