Khim Hoong Chu Eui Yong Kim and Yung Tse Hung

CONTENTS

17.1 Introduction 669

17.2 Description of Horseradish Peroxidase 671

17.3 Model Development 671

17.3.1 Catalytic Cycle and Inactivation of HRP 671

17.3.2 Proposed Reaction Mechanism 672

17.3.3 Derivation of the Reaction Rate Equation 672

17.4 Parameter Estimation and Model Validation 674

17.4.1 Experimental Data 674

17.4.2 Parameter Estimation 674

17.4.3 Model Validation 677

17.5 Model Simulation 677

17.5.1 Dependence of the Reaction Rate on PCP Concentration 677

17.5.2 Dependence of the Reaction Rate on H2O2 Concentration 678

17.5.3 Determination of Optimum H2O2 Concentration 679

Nomenclature 681

Appendix 681

References 684

17.1 INTRODUCTION

Pentachlorophenol (PCP) has been used extensively as a pesticide, herbicide, and wood-preserving agent at many wood treating sites. The chemical structure of PCP is shown in Figure 17.1. It is a probable human carcinogen and has been placed on the U.S. EPA priority pollutant list. Its presence in the environment is therefore of particular concern. In recent years many countries have banned the use of PCP. Unfortunately, past legal disposal practices coupled with the environmental stability of PCP have led to widespread contamination of soil, surface water, and groundwater aquifers. Many of the more than 700 wood preserving sites identified in the U.S. are currently being dealt with under federal, state, or voluntary cleanup programs.1

Various treatment methods can be used to remove PCP from contaminated environmental compartments, and the treatment of PCP-contaminated soil usually involves a combination of physical, chemical, and biological methods. An integrated system combining soil washing with a solvent, recovery of the spent solvent for reuse, and biodegradation of the desorbed PCP in aqueous solution has been proposed.2-4 The biodegradation of aqueous PCP by microorganisms has several advantages over chemical and physical methods, including mild operating conditions and better environmental compatibility. Several species of bacteria and fungi can biodegrade PCP.5-15 These organisms secrete

Handbook of Advanced Industrial and Hazardous Wastes Treatment OH

FIGURE 17.1 Chemical structure of pentachlorophenol (PCP).

a series of oxidative enzymes that are capable of catalyzing the oxidation of PCP. However, high concentrations of PCP can be inhibitory to the activity of the degrading organisms. Cho and colleagues9 have shown that PCP concentrations higher than 50 mg/L inhibit the growth of some PCP-degrading white rot fungi such as Gloeophyllum odoratum and Trametes versicolor completely. As a result, the direct application of isolated enzymes has been proposed as an alternative method of removing PCP from aqueous solution. A number of reviews on the in vitro use of oxidative enzymes to catalyze the oxidation of phenolic substances including PCP are available in the literature.16-18 For PCP oxidation, the enzymes that have been tested include horseradish peroxidase (HRP),19-25 laccase,26-28 ligninase,29 and other extracellular peroxidases.30

Because HRP has been used extensively to transform a wide range of phenolic contaminants, this chapter focuses on the salient aspects of the HRP-catalyzed oxidation of PCP in the presence of hydrogen peroxide (H2O2). The oxidation process generates free aromatic radicals, which combine to form polymers of low solubility that eventually precipitate from solution. Thus, the enzymemediated removal process is also known as polymerization precipitation. The major product of the HRP-catalyzed oxidation of PCP over the pH range 4 to 7 is 2,3,4,5,6-pentachloro-4-pentachloro-phenoxy-2,5-cyclohexadienone (PPCHD).31 PPCHD is formed by the coupling of two pentachloro-phenoxyl radicals, the expected products of one-electron oxidation reactions catalyzed by HRP and other peroxidases. The chemical structure of PPCHD is shown in Figure 17.2. Although the HRPmediated oxidative coupling process has enormous potential for remediation of aqueous solutions contaminated by PCP, its application is hampered by the low operational stability of HRP as a result of inactivation by the enzyme's own substrate, H2O2.32 The key area of interest reported in this chapter is the elucidation of the inhibitory effect of H2O2 on HRP activity. To this end, a theoretical model incorporating saturation kinetics and formation of a catalytically inactive form of HRP in the presence of excess H2O2 was developed to facilitate the quantitative evaluation of the oxidative inactivation of HRP.20 It should be noted that HRP inactivation can occur via two other mechanisms: radical attack and sorption by precipitated products. The analysis of such mechanisms is beyond the scope of this chapter.

Cl Cl Cl Cl

Cl Cl Cl Cl

FIGURE 17.2 Chemical structure of 2,3,4,5,6-pentachloro-4-pentachlorophenoxy-2,5-cyclohexadienone (PPCHD).

17.2 DESCRIPTION OF HORSERADISH PEROXIDASE

As its name implies, HRP (EC 1.11.1.7) is isolated from the roots of horseradish (Armoracia rusticana). A comprehensive description of the structure, function, mechanism of action, and practical applications of HRP has recently been given by Veitch.33 HRP exists in the form of several distinctive isoenzymes, with the C isoenzyme (HRP C) being the predominant form. It consists of 308 amino acid residues, a ferric heme prosthetic group, and 2 mol of calcium per mol of protein, adding up to a molecular weight of 34,520. It is glycosylated and contains four highly conserved disulfide bridges. Recently, there have been key advances in our understanding of HRP and some of these include X-ray crystallographic studies of the crystal structure34 of HRP C as well as the intermediate species in the catalytic cycle of the enzyme.35 HRP can accommodate a broad range of substrates in a variety of reactions. Although it is widely used in analytical diagnostics such as in enzyme immunoassays and biosensors, its low operational stability hampers its commercial applications in organic synthesis for the biotransformations of various drugs and chemicals and in the detoxification of aromatic contaminants.

17.3 MODEL DEVELOPMENT

17.3.1 Catalytic Cycle and Inactivation of HRP

HRP catalyzes the oxidation of a variety of organic and inorganic substances, with H2O2 as electron acceptor. The global reaction catalyzed by HRP is described by Equation 17.1, in which an oxidant (H2O2) reacts with a reducing substrate (AH2) to produce a radical product (•AH) and H2O:

The above reaction proceeds in three distinct steps. First, the native ferric enzyme reacts with the oxidizing substrate (H2O2). Following binding of H2O2 to the heme in the Fe(III) state, the heterolytic cleavage of the oxygen-oxygen bond of H2O2 results in the two-electron oxidation of the heme to form an intermediate (compound I) comprising a ferryl species (Fe(IV) = O) and a prophy-rin radical cation, with the concomitant release of a water molecule. Compound I is a reactive intermediate with a higher formal oxidation state (+5 compared with +3 for the resting enzyme). Compound I is then converted back to the resting enzyme via successive single-electron transfers from two reducing substrate molecules (AH2). The first reduction, of the prophyrin radical cation, yields a second enzyme intermediate, compound II, which retains the heme in the ferryl (Fe(IV) = O) state and the free radical •AH. The second reduction regenerates the ferric heme resting state of the enzyme and delivers another free radical •AH and a water molecule. The catalytic cycle of HRP involving the oxidation and reduction of the heme group can be described by the following reaction scheme:

In these equations, E, Ei, and Eii represent the resting enzyme, compound I, and compound II, respectively.

Numerous studies have shown that oxidation of a wide range of AH2 by HRP in the presence of H2O2 is characterized by a loss of enzyme activity. It is now well established that HRP is inactivated by H2O2.32 Because the final step (Equation 17.4), during which the oxidized ferryl intermediate is reduced, is very slow, inactivation of HRP is thought to occur by reaction of compound II with an additional molecule of H2O2:

where Eiii is known as compound III, which is an inactive form of the enzyme. The degree of inactivation appears to depend on several factors, including the chosen electron donor AH2, the amount of H2O2, and the concentration ratio of H2O2 and the electron donor. The above reaction scheme describes and summarizes the major catalytic and inactivation pathways that have been identified. Because the enzyme is a significant contributor to the cost of contaminant degradation, judicious control of H2O2 concentration to avoid enzyme inactivation will help to enhance the commercial viability of this approach. We describe here a mathematical model that can be used to predict the inhibitory effect of H2O2 on the catalytic behavior of HRP.

17.3.2 Proposed Reaction Mechanism

A kinetic model describing the HRP-catalyzed oxidation of PCP by H2O2 should account for the effects of the concentrations of HRP, PCP, and H2O2 on the reaction rate. To derive such an equation, a reaction mechanism involving saturation kinetics is proposed. Based on the reaction scheme described in Section 17.3.1, which implies that the catalytic cycle is irreversible, the three distinct reactions steps (Equations 17.2 to 17.4) are modified to include the formation of Michaelis-Menten complexes:

In these equations, E*, E*, and E* represent Michaelis-Menten complexes, •P is the PCP-derived radical, k_j, k_3, k_5 and k to k6 are the rate constants of the respective reactions. The existence of the Michaelis-Menten complexes between HRP and H2O2 (E*) and between compound I or compound II and certain reducing substrates (Ei* or Ei*i) has been demonstrated by van Haandel and colleagues,36 Baek and van Wart,37 and Rodriguez-Lopez and colleagues,38 respectively. It should be noted that the radical generation steps in Equation 17.7 and Equation 17.8 have also been proven to be reversible.39 The overall reaction is given by

The radical intermediates •P can couple with each other, leading to the formation of polymeric precipitates that can be readily removed from water (see Figure 17.2). The polymerization of the free radicals is known to be extremely fast, and it is therefore not included in the above reaction scheme.

17.3.3 Derivation of the Reaction Rate Equation

To derive a rate equation based on Equations 17.6, 17.7, and 17.8, the following assumptions are made. First, at the start of the reaction the concentrations of the products are assumed to be zero in comparison with those of the reactants. Thus, these equations can be considered to be essentially irreversible during the early stages of reaction. Second, Equation 17.8 is assumed to be the rate-limiting step, because under most steady-state conditions the reaction of HRP with H2O2 is very fast, and the reaction of compound II with the reducing substrate is at least 10 to 20 times slower than that of compound I.40 The overall reaction rate V is thus given by

Applying steady-state assumptions, the rate equation for the reaction mechanism described by Equations 17.6, 17.7, and 17.8 can be obtained:

k_3 + k4

+

k_5 + k(

k3 k4

k5k6

1

1

1

+

+

k2

k4

k6

k2 k4 k6

where [E0] denotes the initial concentration of enzyme. Further details concerning the derivation of Equation 17.11 by the schematic method of King and Altman41 are given in the Appendix. Equation 17.11 indicates that the reaction mechanism follows the well-known Ping-Pong Bi-Bi mechanism. This mechanism is characterized by the product of the enzyme's reaction with the first substrate (i.e., H2O2), being released before the reaction of the enzyme with the second substrate (i.e., PCP). The general form of the rate equation based on the Ping-Pong Bi-Bi mechanism is given by

where [A] and [B] denote the concentrations of two different substrates, and Kcat, Km, and Km are constants.

Recasting Equation 17.11 in the form of Equation 17.12 gives

V = Krat[ E0HH2O2HPCP]

k 3 + k4

+

k 5 + k

k3k4

k5k6

1

1

1

+

+

k2

k4

k6

k2 k4 k6 k2 k4 k6

Equation 17.13 has been derived without taking account of HRP inactivation by H2O2, which is described in Equation 17.5. One simple way to remedy this situation is to introduce an inactivation constant into Equation 17.13:

[H2O2 K

[H2O2

where Ki is an inactivation constant that describes the inhibitory effect of H2O2. Equation 17.14 may be used to predict the effects of enzyme, H2O2, and PCP concentrations as well as the inhibitory effect of H2O2 concentration on the reaction rate, provided that the four constants Kcat, KmH2O2, KmPCP, and Ki are known. In the next section, we describe how these constants may be estimated by fitting Equation 17.14 to experimental data.

17.4 PARAMETER ESTIMATION AND MODEL VALIDATION 17.4.1 Experimental Data

To generate experimental data for parameter estimation, batch reaction experiments were conducted at 25°C using solutions containing equimolar concentrations of PCP and H2O2 (0.01 to 6 mM) in 100 mM sodium phosphate buffer (pH 6.5). The enzymatic reaction was initiated by adding a dose of HRP stock solution to the reaction mixture. Experiments were conducted at four different initial enzyme concentrations: 0.13, 0.148, 0.295, and 0.34 |M. Solution samples were taken at fixed time intervals and centrifuged to settle precipitated colloidal particles. The PCP concentration of the supernatant was determined using a UV spectrophotometer. Initial reaction rates were estimated from the initial slopes of PCP concentration versus time curves. Additional experiments were conducted to generate a new set of data for model validation. In these experiments, the initial enzyme and PCP concentrations were fixed at 0.72 ||M and 1.5 mM, respectively, while the initial H2O2 concentration was varied in the range 0.01 to 12 mM.

17.4.2 Parameter Estimation

The four constants in Equation 17.14 may be estimated by fitting the equation to the measured initial reaction rate data presented in Figure 17.3. Because equimolar concentrations of the two substrates, PCP and H2O2, were used in the experiments, Equation 17.14 may be simplified as follows:

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