Immobilization of horseradish peroxidase in Ca-alginate beads: Evaluation of the enzyme leakage on the overall removal of an azo-dye and mathematical modeling

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Abstract

Horseradish peroxidase (HRP) was immobilized in calcium alginate beads by extruding an alginate/HRP solution through a syringe onto calcium chloride. The aim of this study was to evaluate their use to remove the azo-dye Orange II (OII), under different experimental conditions, such as the number of beads, pH, reuse of the biocatalyst, hydrogen peroxide feeding strategy. A mathematical model that takes into account the diffusion of reactants, products, and HRP throughout the beads and the effect of the enzyme leakage on the observable oxidation rate was also developed. Results indicated that the immobilization efficiency decreased as a function of the incubation time in CaCl2. Although enzyme-containing beads had a low OII adsorption capacity, under the presence of hydrogen peroxide, the dye removal was highly increased due to the catalytic activity of HRP. Besides, the initial OII oxidation rate (RS0) using 3 beads at pH 9 was higher than at pH 7. Also, RS0 increased as a function of the number of tested beads. The developed model was fitted to experimental data obtained at different conditions. Then, the model was validated against a new data set that was not used for the calibration of the model, obtaining a satisfactory agreement between simulation results and these new data. Simulations demonstrate that 70–90% of the total OII removed was due to its oxidation by hydrogen peroxide catalyzed by HRP in the liquid phase.

Introduction

From the green chemistry view, enzymes have become increasingly important in several industries, such as the production of fine chemicals, food processing, pharmaceuticals, and wastewater treatments, among others (Hoyos et al., 2017). In this sense, the use of enzymes is a simple and eco-friendly approach to obtain high reaction rates under predictable conditions. Also, in comparison with physicochemical or microbiological methods, enzyme-catalyzed reactions could be a cost-competitive alternative (Agarwal et al., 2016). However, the main drawbacks concerning the use of enzymes are related to the long-term operational stability, recovery, and reuse. In this sense, several authors report that immobilization of enzymes increases their stability in aqueous media, and also allows their reusability, favoring the development of economically feasible bioprocesses (Wang et al., 2016, Bilal et al., 2018).

As a general rule, immobilization techniques can be classified depending on the physical (e.g., adsorption) or chemical (e.g., covalent bonding, cross-linkage, entrapment) method used for immobilization. Chemical methods are usually preferred in the case of highly cost enzymes. In these methods, the matrix is of vital importance to determine the function and use of the immobilized enzyme (Sneha et al., 2019). In recent years, several authors studied the chemical immobilization of peroxidases onto nanofibrous matrices (Xu et al., 2015, Morales Urrea et al., 2021), nanofibers blending with carbon nanotubes (Zhang et al., 2014), and Buckypaper/polyvinyl alcohol nanocomposite membranes (Jun et al., 2019, Jun et al., 2020a, Jun et al., 2020b).

Among available immobilization techniques, entrapment of enzymes within a polymer matrix, such as calcium alginate, is one of the simplest immobilization methods (Sofia et al., 2016, Sneha et al., 2019). Briefly, a solution of sodium alginate and the enzyme is added drop-wise to a calcium solution. Then, an instantaneous interfacial polymerization produces beads with the immobilized enzyme inside. As calcium ions permeate through the beads, more gelation occurs, increasing the bead hardness. As a general rule, beads diameter and porosity, degree of enzyme leakage, and mechanical stability of the beads can be optimized by the adjustment of different parameters as alginate and calcium concentrations, contact time in the calcium solution, and the use of cross-linking agents (Sneha et al., 2019). In recent years, this technique was widely used to immobilize several peroxidases, such as manganese peroxidase (Bilal and Asgher, 2015), lignin peroxidase (Bilal and Iqbal, 2019), and horseradish peroxidase (Bilal et al., 2016a, Bilal et al., 2017, Farias et al., 2017).

Sneha et al. (2019) report that one of the main problems regarding the immobilization of enzymes in Ca-alginate beads is their leakage. As a general rule, beads gradually release the entrapped enzyme to the solution as a function of time. For this reason, the observable reaction rate not only depends on the reaction rate within beads but also in the aqueous phase. Accordingly, measured reaction rates may also depend on the enzyme leakage rate. Enzyme leakage can be reduced, but not avoided, by the use of cross-linking agents, such as glutaraldehyde (Bilal et al., 2016a), or polyvinyl alcohol (Bilal et al., 2017), or by the addition of compounds that reduce the diffusion of the enzyme in the beads, such as bentonite (Rodriguez et al., 2018) or boehmite (Ai et al., 2013). However, up to the present, there is no available information concerning the role of the leakage process on the observable reaction rate. In the present study, horseradish peroxidase (HRP) was immobilized in Ca-alginate beads and used to remove the azo-dye Orange II as a model compound. Experimental conditions, such as the number of beads, pH, reuse of the biocatalyst, hydrogen peroxide feeding strategy, were evaluated. Then, a mathematical model that takes into account the effect of the enzyme leakage on the observable oxidation rate was developed.

Section snippets

Chemicals and reagents

Orange II (OII) sodium salt (purity ≥ 85%, CAS Number ‎633-96-5), calcium chloride (CaCl2) (anhydrous, powder, purity ≥ 97%, CAS Number 10043-52-4), and horseradish peroxidase (HRP) Type I (CAS Number 9003-99-0) were purchased from Sigma Aldrich. The enzyme was supplied as a lyophilized powder. According to the manufacturer, the specific activity was 146 units/mg of the solid powder (one unit corresponds to the amount of enzyme that forms 1 mg of purpurogallin from pyrogallol in 20 s at pH 6

Immobilization of HRP in Ca-alginate beads

Horseradish peroxidase (HRP) was immobilized in calcium alginate beads by extruding an alginate/HRP solution through a syringe onto calcium chloride. The number of beads per unit volume of alginate/HRP extruded solution (nv) obtained in 20 immobilization assays ranged between 30 and 100 beads/mL. For each experiment, the spherical equivalent bead diameter (dB) was calculated using Eq. (1). A Shapiro–Wilk normality test (Shapiro and Wilk, 1965) demonstrated that the distribution corresponding to

Conclusions

In this work, horseradish peroxidase (HRP) was immobilized in calcium alginate beads (AHB) by extruding an alginate/HRP solution through a syringe onto calcium chloride. The effect of several experimental conditions, such as the contact time in CaCl2, the number of beads, pH, reuse of the biocatalyst, and hydrogen peroxide feeding strategy, on the removal of the model compound Orange II (OII) using the obtained AHB were studied. Although AHB had a low OII adsorption capacity, the dye removal

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors wish to acknowledge the National Agency for Scientific and Technological Promotion (ANPCyT), and the National Council for Scientific and Technical Research (CONICET) for financial support.

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