Entrapment of enzyme in the presence of proline: effective approach to enhance activity and stability of horseradish peroxidase

Abstract

In this report, activity and stability of horseradish peroxidase (HRP) entrapped in polyacrylamide gel in the presence of proline (HEP) are compared with that of enzyme entrapped in absence of proline (HE). Within polyacrylamide (8%) beads, 80% entrapment yield for peroxidase was observed in the presence as well as absence of proline. The HEP (1.5 M proline) showed 170% higher activity compared to HE. HEP also showed significant increase in K_M, V_max and K_cat. At 8th cycle of use, HEP retained 40% of its activity, whereas HE retained only 10% of activity. In addition, in comparison with HE, HEP showed increased storage stability and thermo-stability. HEP showed higher activity compared to HE over an extensive range of pH (4–8), temperature (30–80 °C) and inhibitors such as NaN₃, Cd²⁺ and Pb²⁺. Our results suggest that peroxidase entrapment in polyacrylamide gel in the presence of proline can be a useful approach for increasing activity and stability of horseradish peroxidase.

Introduction

Peroxidases belong to oxido-reductase class of enzyme and catalyze the oxidation of various aromatic compounds. They have broad applications in many areas including chemical synthesis, diagnostic, medicine, pulp and paper industries, food processing industry and remediation of wastewaters (Agostini et al. 2002; Moreira et al. 2007; Bansal and Kanwar 2013; Chiong et al. 2014; Agarwal et al. 2018). Aromatic compounds including phenols, cresols, and chlorinated phenols are the major pollutants present in wastewaters of various industries including petroleum refining, coal conversion and pulp and paper industries (Mojiri et al. 2019). Removal of these pollutants from wastewaters is of prime importance due to their high toxic effect in living organisms. Over past decades, extensive research has been carried out for removal of contaminants from water. Application of enzymes has been found to be a potential method for successful removal of contaminants over conventional methods. Advantages of using enzymes for wastewater treatment are their ability to operate over a broad concentration range of substrate and requirement of low retention times (Karam and Nicell 1997; Unuofin et al. 2019). Peroxidase obtained from horseradish (HRP) is used for the laboratory-scale treatment of aqueous aromatic contaminants due to its broad substrate specificity (Bayramoglu and Arıca 2008; Alemzadeh and Nejati 2009). However, free enzyme may suffer from several limitations such as high price and inadequate stability due to different conditions (Li et al. 2018). The change in reaction conditions and presence of denaturizing agent disrupt the performance and hamper the activity of enzymes (Veitch 2004). Heavy metals such as Cd²⁺, Pb²⁺ and NaN₃ are usually present as pollutant along with aromatic compounds and have the capability to inhibit the activity of peroxidase (Mahmoudi et al. 2003); therefore, they present challenges in the wastewater treatment industry.

Immobilization of enzyme proves to be an efficient tool for overcoming the limitations of free enzymes (Husain and Ulber 2011). Immobilization not only makes enzyme reusable thus reducing overall cost but also enhances its activity, specificity, selectivity, stability and performance under specific conditions (Mohamad et al. 2015; Fernandez-Lafuente 2009). However, enzymes may encounter some denaturing stresses during immobilization. Immobilized enzyme suffers from distorted protein configuration, steric hindrance and slow diffusion rate between the immobilized surface and substrate (Vineh et al. 2018). Compatible solutes which are low molecular weight inert molecules are suggested to protect enzymes in vitro (Xie and Timasheff 1997; Sharma and Dubey 2004, 2005; Mishra and Dubey 2006; Jha and Sharma 2019). Proline is one of the most studied compatible solutes that can stabilize protein (Sharma and Dubey 2004, 2005; Bozorgmehr and Monhemi 2015; Jha and Sharma 2019). Although entrapment of enzymes in the presence of several additives has been conducted, there is no report of enzyme entrapment in the presence of proline. Therefore, the present study was focused on to examine the activity, stability and reusability of the enzyme peroxidase entrapped in polyacrylamide gel in the presence of proline (HEP) and compare it with that of peroxidase enzyme entrapped in the absence of proline (HE). Furthermore, the protective effects of proline on peroxidase enzyme against metal inhibitors like Cd²⁺, Pb²⁺ and NaN₃ often present in wastewater along with aromatic compounds were also studied.

Materials and methods

Peroxidase from horseradish (Sigma Type VI) was purchased from Sigma-Aldrich (St Louis, MO, United States). All other chemicals of analytical grade were purchased from Himedia or Sigma, Mumbai, India.

Immobilization of peroxidase in polyacrylamide gel beads

Entrapment of HRP was carried out by polymerizing polyacrylamide gel in the presence of 20 U HRP with (0.5, 1.0 and 1.5, 2, 2.5, 3.0 M) or without (0 M) proline. To study the effect of different concentrations of acrylamide on peroxidase entrapment, polymerization was performed by mixing 4.0, 6.0, 8.0 and 10% acrylamide and N,N′-methylene bisacrlyamide (30:0.8%) solution with HRP (20 U), 500 µl potassium phosphate buffer (1 M), 200 µl ammonium persulfate, 50 µl tetramethylethylenediamine (TEMED) and double deionized water to make total volume 10 ml. The resulting mixture was immediately poured uniformly in 10 cm diameter petri plates and allowed to polymerize for 90 min at room temperature. Gel was then cut into equal pieces of approximately 2 mm size and then washed thrice with distilled water to remove any free enzymes present. For further use, gel pieces were air dried and stored at 4 °C. Each gel was stored maximum for 30 days at 4 °C. All the experiments were conducted in triplicate and the results are the mean ± SD values of three observations.

Peroxidase activity assay

The activity of free and HRP entrapped in polyacrylamide gel beads was determined according to the method of Egley et al. (1983) with few modifications. The assay mixture consisted of 50 mM potassium phosphate buffer (pH 7.0), 2 mM H₂O₂, 9 mM guaiacol and 50 µl of supernatant or 50 mg gel piece in case of entrapped enzyme in total volume of 3 ml. The change in absorbance at 470 nm was recorded at 30 s intervals for 3 min using a Thermo Scientific 10S UV–Vis spectrophotometer for tetra guaiacol formation. The relative activity was calculated in percentage with reference to the activity of enzyme entrapped in the absence of proline (100%) using equation:

$$\text{Relative}\text{peroxidase}\text{activity}\left(\%\right)=\frac{\text{activity}\text{of}\text{entrapped}\text{peroxidase}\text{in}\text{presence}\text{of}\text{proline}\times 100}{\text{activity}\text{of}\text{entrapped}\text{peroxidase}\text{in}\text{absence}\text{of}\text{proline}}.$$

Immobilization yield

The immobilization yield of peroxidase entrapped in the presence and absence of proline was calculated using equation:

$$\text{Immobilization}\text{yield}\left(\%\right)=\frac{\left(\text{activity}\text{of}\text{total}\text{peroxidase}\text{used}\text{for}\text{immobilization}-\text{activity}\text{of}\text{peroxidase}\text{in}\text{supernatant}\right)\times 100}{\text{activity}\text{of}\text{total}\text{peroxidase}\text{used}\text{for}\text{immobilization}}$$

Determination of V_max, K_M and K_cat

The values of V_max and K_M for guaiacol were obtained using different concentrations of guaiacol (0.5, 1, 2, 3, 4 mM) and keeping H₂O₂ concentration fixed at saturating value 2 mM. The V_max and K_M values were calculated using linear regression analyses of the data points of double-reciprocal plots (Lineweaver and Burk 1934). Turnover number (K_cat) (Garrett and Grisham 1999) was calculated using equation:

$$K_{\text{cat}}=\frac{V_{max}}{\text{Et}},$$

where Et is the total enzyme concentration.

Reusability and storage stability of entrapped peroxidase

Effect of proline on reusability of entrapped peroxidase enzyme for 15 cycles was investigated. For each cycle, the activity of entrapped peroxidase was checked after washing the gel piece two times with double distilled water. The relative activity was calculated in percentage with reference to the activity obtained in the first cycle (100%). Storage stability of HRP enzyme immobilized in the presence and absence of proline at 4 °C was determined by measuring enzyme activity every 5 days up to 30 days.

Effect of pH and temperature on entrapped peroxidase activity

The effect of pH on the activity of entrapped peroxidase in the presence and absence of proline was evaluated at different pH ranging from 4.5 to 8. Calculation of the relative activity was done with reference to the activity of HEP (1.5 M Proline) at optimum pH. The optimum temperature and thermal stability of peroxidase enzyme entrapped in the presence and absence of proline was evaluated at different temperatures (30 °C, 40 °C, 50 °C, 60 °C, 70 °C and 80 °C). Enzyme assay mixture containing 50 mg gel pieces were placed in water bath at different temperature for 10 min. Furthermore, it was allowed to cool back to room temperature for 30 min and peroxidase activity was assayed. Calculation of the relative activity was done with reference to the activity at optimum temperature (100%).

Effect of NaN₃, Cd²⁺and Pb²⁺ on entrapped peroxidase activity

The effects of NaN_3, Cd²⁺ and Pb²⁺ on the activity of entrapped peroxidase in the presence and absence of proline were investigated. For this, 50 mg gel pieces of entrapped enzyme were incubated with NaN₃ concentration of 1 and 5 mM and Cd²⁺ and Pb²⁺ concentrations of 250 and 500 µM. Furthermore, the change in activity was recorded as described above at 470 nm. The relative activity is expressed as a percentage of the original activity assayed without the metal ions (100%).

Statistical analysis

All the experiments were performed in triplicate. Values indicate mean values ± SD. Data were analysed by one-way analysis of variance (Tukey’s multiple-comparison test) using Graphpad Prism 6.0 software to test statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001).

Results and discussion

Many studies showing the effect of protein stabilizing additives on enzyme activity have been reported (Brennan et al. 2003; Kalaiarasan and Palvannan 2015). However, to the best of our knowledge, no report is available regarding evaluation of the activity and stability of enzyme immobilized in the presence of proline. Peroxidases have wide application in various industries and remediation of wastewaters (Moreira et al. 2007; Bansal and Kanwar 2013; Chiong et al. 2014; Agarwal et al. 2018). In the present study, we assessed the activity and stability of HRP entrapped in polyacrylamide gel beads in the presence of proline and compared them with that of HRP entrapped in the absence of proline. Effect of different concentration of acrylamide (5.0, 6.0, 7.0, 8.0 and 9.0, 10.0, 11.0, 12.0%) was studied on the entrapment of peroxidase. The 8% polyacrylamide beads exhibited maximum entrapment yield for peroxidase (Fig. 1a). Peroxidase enzyme entrapped in polyacrylamide (8.0%) gel bead in the presence as well as absence of proline showed 80% entrapment efficiency. Effect of proline on enzyme activity was concentration dependent (Fig. 1b). Entrapment in the presence of 0.5, 1.0 and 1.5 M proline led to 50, 100 and 170% higher enzyme activity compared to entrapment in the absence of proline (Fig. 1b). At high concentrations (> 1.5 M), proline forms loose amphipathic higher-order molecular aggregates (due to stacking) which can decrease the hydrophobicity of the proteins and provide stability/protection against denaturation encountered during entrapment process (Schobert and Tschesche 1978; Samuel et al. 2000). Paleg et al. (1984) performed experiments to see whether proline can provide protection at lower concentrations (in the range 0.03–1 M) at which it does not possess stacking ability. They observed concomitant increase in protective effect with increasing concentration of proline which can be attributed to the conformation changes and alteration in the hydration of entrapped protein (Brennan et al. 2003; Kalaiarasan and Palvannan 2015; Kossowska et al. 2018). Previously, other additives like carbohydrates, dextran, sorbitol and bovine serum albumin have also been shown to increase the activity of entrapped enzymes (Altikatoglu and Basaran 2011; Ni et al. 2013; Bibi et al. 2015; Kondyurin et al. 2015). The kinetic parameters K_M and V_max for enzyme entrapped in the absence and presence of 1 and 1.5 M proline were determined (Fig. 2a–c). The calculated K_M and V_max values of HE, HRP entrapped in the presence of 1 M proline and HRP entrapped in the presence of 1.5 M proline were 0.69 mM and 69.4 nmoles min⁻¹ mg⁻¹, 1.6 mM and 147.0 nmoles min⁻¹ mg⁻¹ protein and 2.0 mM and 212.7 nmoles min⁻¹ mg⁻¹ protein, respectively. Our results indicated significant increase in K_M as well as V_max of peroxidase enzyme entrapped in the presence of 1 or 1.5 M proline. Around 2.3 fold increase in K_M and 2.1 fold increase in V_max value of peroxidase were observed in the presence of 1 M proline, whereas around 2.9 fold increase in K_M and 3.0 fold increase in V_max was observed in the presence of 1.5 M proline. The turnover number calculated for HE, HEP (1 M) and HEP (1.5 M proline) were 2430.4 s⁻¹, 5146.7 s⁻¹ and 7420.0 s⁻¹, respectively. In general, K_M indicates the affinity between substrate and enzyme and thus, among important kinetic parameter. Dissociation of product is also one of the factors important for enzyme activity. Increase in K_M (less affinity of products for enzyme) as well as V_max in the presence of proline could be the reason for high turnover number shown by HEP (1 and 1.5 M proline).

Fig. 1.

Immobilization yield of peroxidase at different acrylamide concentration (a) and relative activity of horseradish peroxidase immobilized in 8% polyacrylamide gel beads in the presence of 0.5, 1 M and 1.5 M proline with reference to the activity of enzyme entrapped in the absence of proline (100%) (b). Values are mean ± SD based on three independent determinations and bars indicate standard deviations. *, **, and *** represent significant differences in peroxidase activity compared to activity of HRP immobilized in 5% polyacrylamide gel bead (a) and HRP entrapped in the absence of proline (b) at P < 0.05, P < 0.01 and P < 0.001, respectively

Fig. 2.

Double reciprocal plots for peroxidase entrapped with 0 M (a) 1 M (b) and 1.5 M (c) proline using guaiacol as substrate. Values are the mean of three independent determinations

Storage stability, a key factor for commercialization of an enzyme was evaluated for HRP enzyme immobilized in the presence and absence of proline at 4 °C. HEP showed 100% activity up to 30 days when stored at 4 °C. Residual activity of 75% was observed for HE on the 30th day at 4 °C (Fig. 3a). One of the major advantages of using immobilized enzyme in industrial processes is its reusability. Therefore, the effect of proline on reusability of entrapped enzyme was investigated up to 15 cycles. HEP (1.5 M proline) retained 40% activity at 8th cycle compared to 10% retention of peroxidase activity in the absence of proline (Fig. 3b). In both cases, relative activity was estimated with reference to the activity of entrapped enzyme present in first cycle. The effect of pH and temperature on activity of peroxidase entrapped in the presence and absence of proline was determined by incubating entrapped enzymes at different pH (4.5–8) and temperatures (30–80 °C) (Fig. 4a, b). HEP showed higher activity compared to HE at all the pH and temperature studied; however, optimum pH and temperature were same in both the cases. At 80 °C, enzyme entrapped in the presence of 0 M and 1.5 M proline showed relative activity of 34% and 54%, respectively, with reference to the activity at optimum temperature which was 40 °C for both the cases indicating proline led thermostability of peroxidase enzyme (Fig. 4b). Protein stability in aqueous solutions of osmolytes is a balance between the preferential hydration of solutes (proteins), preferential exclusion of osmolytes from the protein surface, and the osmolyte binding to the protein surface (Bruździak et al. 2015). Thermodynamic stabilization of the protein in the presence of proline has been suggested due to increase of water molecules in the hydration shell around the protein. In addition, molecular aggregates of proline in the solution can increase the order of water molecules and also the water–water hydrogen bond strength leading to enhanced thermal stability of the protein (Bozorgmehr and Monhemi 2015). Increased thermostability of peroxidase enzyme offers advantage due to its ability to withstand elevated temperatures.

Fig. 3.

Relative activity at different days of storage of HRP entrapped with 0 or 1.5 M proline at 4 °C with reference to the activity of HRP on first day (a) and at different reuse cycle with reference to the activity of HRP at first cycle (100%) (b). Values are mean ± SD based on three independent determinations and bars indicate standard deviations. ***Significant differences in peroxidase activity compared to activity of HRP immobilized in the absence of proline at P < 0.001

Fig. 4.

Relative activity of HRP immobilized in the presence and absence of 1.5 M proline at different pH with reference to the activity of HRP immobilized in the presence of 1.5 M proline at optimum pH (a) and at different temperature (b) with reference to the activity of enzyme entrapped in the presence of 1.5 proline at optimum temperature (40 °C). Values are mean ± SD based on three independent determinations and bars indicate standard deviations. ***Significant differences in peroxidase activity compared to activity of HRP immobilized in the absence of proline at P < 0.001

Peroxidases can be used in remediation of wastewater contaminated with hazardous aromatic compounds (Na and Lee 2017; Singh et al. 2017; Zheng et al. 2019). Sodium azide (NaN₃), a toxic compound and heavy metals such as Cd²⁺_, Pb²⁺ are usually present as co-contaminant and can inhibit peroxidase activity at high concentrations (Mahmoudi et al. 2003; Singh et al. 2017). NaN₃ treatment led to 27 and 59% decrease in HE activity but only 10 and 26% decrease in HEP activity at 5 mM and 10 mM, respectively (Table 1). Metal ions may coordinate to active-site residues of enzymes and activate it; however, such coordination may lead to inhibition due to blockage of substrate interaction site. At low concentrations, metals have been shown to activate enzyme, whereas at higher concentrations, they inhibit enzyme activity (Shi et al. 1992; Singh et al. 2017). Proline has capability to stabilize proteins and enzymes. Therefore, the inhibitory effects of increasing concentrations of Cd²⁺ and Pb²⁺ on HEP and HE were compared. HEP showed higher activity in the presence of Cd²⁺ and Pb²⁺ with reference to HE activity. Pb²⁺ and Cd²⁺ treatments of 250 µM led to enhanced HEP but decreased HE activity (Table 1). Incubation with 250 µM Pb²⁺ and Cd²⁺ led to 22 and 14% increase, respectively, in HEP activity but decrease of 13% and 19% in HE activity (Table 1). Incubation with 500 µM Pb²⁺ and Cd²⁺ concentration led to only 7% and 10% decrease, respectively, in HEP activity but 29% and 32% decrease in HE activity. Results showed that the proline entrapped along with peroxidase in polyacrylamide gel confer protection against metal denaturation. This could be explained by the capability of proline to reduce metal by forming metal-proline complex (Sharma et al. 1998).

Table 1.

Effect of different inhibitors (NaN₃, CdCl₂, PbCl₂) on the relative activity of horseradish peroxidase entrapped in the presence and absence of 1.5 M proline with reference to control (100%)

Treatment	Concentration	Relative activity of entrapped horseradish peroxidase (%)
		0 M proline	1.5 M proline
Control	0	100.00 ± 5.30	100.00 ± 6.25
NaN3	5 mM	73.07 ± 5.23*	90.16 ± 4.75
	10 mM	41.31 ± 6.67**	74.76 ± 7.53*
CdCl2	250 µM	81.57 ± 8.18	114.62 ± 4.23
	500 µM	68.15 ± 3.25**	89.91 ± 5.65
PbCl2	250 µM	87.63 ± 7.23	121.90 ± 7.45
	500 µM	71.31 ± 6.28*	93.00 ± 5.96

Values are mean ± SD based on three independent determinations and bars indicate standard deviations

* and ** represent significant differences compared to control at P < 0.05 and P < 0.01, respectively

Conclusions

Overall, our results show that the inclusion of proline during immobilization leads to significant improvement in activity, reusability and stability of HRP enzyme within polyacrylamide gel beads. Present study showed optimum percentage of polyacrylamide gel beads (8%) and concentration of proline (1.5 M). HEP showed higher activity compared to HE at all the pH and temperature studied and retained 40% activity at 8th cycle compared to 10% retention of peroxidase activity in the absence of proline. This study shows that various factors necessary for commercialization of an enzyme were improved by entrapment in the presence of proline. HEP showed lesser peroxidase inhibition compared to HE due to high concentrations of NaN_3, Cd²⁺ and Pb²⁺ which are usually present in wastewater. Further studies are required to apply this promising approach in wastewater treatment and various industries.

Abbreviations

HE

Horseradish peroxidase enzyme entrapped in the absence of proline

HRP

Horseradish peroxidase enzyme

HEP

Horseradish peroxidase enzyme entrapped in polyacrylamide gel in the presence of proline

K_cat

Turnover number, a constant which depicts the rate of turnover of an enzyme–substrate complex into product

K_M

Michaelis constant, a constant which depicts substrate concentration that allows the enzyme to attain half V_max

V_max

Maximum rate of reaction, reaction rate when the enzyme is saturated with substrate

Compliance with ethical standards

Conflict of interest

There is no conflict of interest related to this study.