TITLE THERMODYNAMICS AND KINETICS OF THERMO-INACTIVATION AND REGENERATION OF PARTIALLY PURIFIED PEROXIDASE FROM Gongronema latifolium LEAVES

ABSTRACT

Peroxidase activity from G. latifolium was done to see whether it could be used in industries. Crude peroxidase was extracted from G. latifolium with 0.05M sodium phosphate buffer of pH 6.0; 70% ammonium sulphate saturation to precipitated protein with the highest G. latifolium peroxidase activity. After gel filtration, two major peaks were seen and the active fractions were pooled differently together and characterized. The optimal pH for the enzyme peaks actually were found to be 6.5 and 6.0 and the optimum temperature was 30 and 40ºC for peak A and B respectively. The Michealis-Mentenconstant (K_m) and maximum velocity (V_max­) obtained from the Lineweaver-Burk plot of initial velocity of different substrate concentration were found to be 1.242 mM and 20.83 U/min for hydrogen peroxide concentration [H₂O₂] and 0.109 mM and 10.99 U/min for o-dianisidine concentrations. On the thermal stability assessment of the enzyme. Thermal inactivation profiles of these enzyme peaks follows first order kinetics with the time required varying with the product of the studies. The half-lives of the enzyme at the two peaks were obtained to be 770.16 mins at 30ºC  for peak A and 330.07 mins at 40ºC  for peak B, the activation energy for inactivation (E_a(inact)) calculated from the Arrhenius plot were found to be 67.55 KJmol^-1 and 59.58 KJmol^-1 forpeaks A and B, respectively. The Z-values were obtained to be 30.21 and 34.25 for the two enzyme peaks respectively. The thermodynamics parameters obtained for the two enzyme peaks were as follows: change in enthalpy of inactivation (ΔH_(inact)) 65.026 KJmol^-1K^-1 for peak A at 30ºC and 56.982 KJmol^-1K^-1 at 40ºC for peak B; the change in free energy of inactivation, (ΔG_(inact)) values for the two enzyme peaks were 102.229 KJmol^-1K^-1  at 30 ºC and 103.483 KJmol^-1K^-1  at 40ºC for peak A and B respectively. The entropy of inactivation (ΔS_(inact)) values for the two enzyme peaks were calculated to be -0.1228 KJmol^-1K^-1 at 30ºC and -0.149 KJmol^-1K^-1 at 40ºC. Reactivation of the Gongronema latifolium peroxidase occurred rapidly, within first 30 minutes after the heated enzyme was cooled and incubated at room temperature. The extent of reactivation varied from 0 to 20% depending on the isoenzyme and heating conditions (temperature and time). The denaturation temperature allowing the maximum reactivation was 50°C and 40°C for peaks A and B respectively. In all cases, heat treatment at high temperatures for a long period prevented reactivation of the heated enzymes. The peak A peroxidase regained activity rapidly, within 30 minutes at 30 and 40°C and within 60 minutes at 50, 60, 70 and 80°C after the heated enzyme was cooled and incubated at room temperature. However, peak B peroxidase regained activity rapidly within 60 minutes at all the study temperature after the heated enzyme was cooled and incubated at room temperature.The kinetic and thermodynamic parameters and higher activation energies from this study suggest that this enzyme could be more suitable for several industrial applications.

CHAPTER ONE

INTRODUCTION

The super-family of haem peroxidases from plants, fungi and bacteria is a group of enzymes that utilize hydrogen peroxide to oxidize a second (reducing) substrate often aromatic oxygen donor. These enzymes share similar catalytic cycles where hydrogen peroxide reacts with the resting ferric enzyme to form the intermediate compound I (known as compound ES in cytochrome c peroxidase) which carries two oxidizing equivalents. Compound I is subsequently reduced by reaction with two reducing substrate molecules. The reaction of these reduction steps generate the intermediate, compound II, which is then further reduced back to the ferric enzyme (Hiner et al.,2000). Peroxidase forms part of the defense system of living organisms against radical-mediated peroxidation of unsaturated lipids. They are ubiquitous in nature and are involved in various physiological processes in plants. Studies have suggested that peroxidases play a role in lignification, suberization, cross-linking of cell wall structural protein, auxin catabolism and self –defense against pathogens and senescence (Hiraga et al., 2001). Currently, industrial application of peroxidase in chemistry, pharmacology and biotechnology is well developed. Peroxidase is used in waste treatment in order to remove aromatic phenols and amine from aqueous solution in the presence of hydrogen peroxide. In this treatment, phenolic compounds are polymerized in the presence of hydrogen peroxide through a radical oxidation-reduction mechanism (Nazari et al., 2005). As hydrogen peroxide concentration increases, an irreversible mechanism-based inactivation process becomes predominant (Rodriguez-Lopez et al., 1997)  and it leads to the degradation of haem, the release of iron and the formation of two fluorescent products (Gutteridge, 1986). Different agents like temperature and chemicals promote enzyme inactivation. Temperature produces opposed effects on enzyme activity and stability and it is therefore a key variable in any biocatalytic process (Wasserman, 1984). Biocatalyst stability i.e the capacity to retain activity through time is undoubtedly the limiting factor in most bioprocesses, biocatalyst stabilization being the central issue of biotechnology (Illanes, 1999). Biocatalyst thermostability allow a higher operation temperature, which is clearly advantageous because of higher reactivity, higher process yield, lower viscosity and fewer contamination problem (Mozhaev, 1993). Enzyme thermal inactivation is the consequence of weakening the intermolecular forces responsible for the preservation of its 3D-structure, leading to a reduction in its catalytic capacity (Misset, 1993). Inactivation may involve covalent or non-covalent bond disruption with subsequent molecular aggregation or improper folding (Bommarius and Broering, 2005). Knowledge on enzyme inactivation kinetics under process condition is an absolute requirement to properly evaluate enzyme performance (Illanes et al., 2008).

 

1.1       Peroxidase

Peroxidase (EC1.11.1.7) an antioxidant enzyme is widely distributed in microbes, plants, and animal tissues and represents a haem-containing enzymes family (Huystee and Cairns,1982). It has been widely reported that the presence of residual endogenous enzymes in both raw and processed fruits and vegetable products may cause loss of quality during storage (Anthon and Barrett, 2002; Chilaka et al., 2002). This oxidoreductase catalyzes a reaction in which hydrogen peroxide acts as acceptors and another compound acts as the donor of hydrogen atoms (Rodrigo et al., 1996; Kvaratskheliaet al., 1997). In the presence of peroxide, peroxidase from plant tissues are able to oxidize a wide range of phenolic compounds, such as o-dianisidine dichlororide, guaiacol, catechol, pyrogallol, chlorogenic acid, and catechin (Onsa et al., 2004). This enzyme can provide value for multiple industrial applications. Of the most important ones include decoloration of dye waste (Jadhav et al., 2009), treatment of waste water containing phenolic compounds (Lia and Lin., 2005) and synthesis of various aromatic chemicals and removal of peroxide from food stuffs and industrial wastes (Saitou et al.,1991; Kim and Yoo, 1996). Peroxidases, ubiquitously existing in nature are divided into three groups according to their amino acid sequences: class I (ascorbate type), class II (fungal secretary) and class III (guaiacol type, plant secretary) (Das et al., 2011). Most of the plant secretory peroxidases are glycosylated proteins (Johansson et al., 1992; Kvaratskhelia et al., 1997). They play critical roles in physiological functions such as lignification, cell wall metabolism, enhancing plant resistance, eliminating H₂O₂ which injures and participates in auxin catabolism (Lagrimini, 1996;Hiraga et al., 2001;Boka and Orban, 2007). It was reported that plant secretory peroxidases had been purified and characterized from lots of plants such as horseradish, soybean, bitter gourd and tea (Kvaratskhelia et al., 1997; Lavery et al., 2010). Peroxidase from horseradish (Aromoracia rusticana) are commonly used in pharmaceutical industry, waste water treatment, bio-sensor construction and food industry (Jia et al., 2002;Koksal and Gulçin, 2008;Lavery et al., 2010). Horseradish peroxidase is a haem glycoprotein which is isolated from the root of horseradish like garlic, ginger e.t.c. It is able to oxidize a wide scale of substrates using hydrogen peroxide as the oxidizing agent. In its native state, the enzyme contains a ferric (Fe³⁺) protoporphyrin IX as prosthetic group where Fe³⁺ is coordinated to a (proximal) histidine (Koksal and Gulcin, 2008). Numerous studies have been carried out in a search of an alternative source of peroxidase with higher stability, availability, degree of purification, and substrate specificity.

 

1.1.1    Functional roles

Most reactions catalysed by peroxidase especially horseradish peroxidase can be expressed by the following equation, in which AH₂ and ⁰AH represent a reducing substrate and its radical product respectively. Typical reducing substrates include aromatic phenols, phenolic acids, indoles, amines and sulfonates.

H₂O₂ + 2AH₂ + POD → 2H₂O + 2⁰A…………………………………………………..Reaction(I)

Figure 1: Catalytic cycle of peroxidase (Villalobos and Buchanan, 2002).

During the catalytic cycle of peroxidase as shown in figure 1, the ground state enzyme undergoes a two electron oxidation by H₂O₂ forming an intermediate state called compound I (E). Compound I (E) will accept an aromatic compound (AH₂) in its active site and will carry out its one-electron oxidation, liberating a free radical (⁰AH) that is released back into the solution, converting compound I (E) to compound II (Ei). A second aromatic compound (AH₂) is accepted in the active site of compound II (Ei) and is oxidized, resulting in the release of a second free radical (⁰AH) and the return of the enzyme to its resting state, completing the catalytic cycle (Figure 1). The two free radicals (⁰AH) released into the solution combine to produce insoluble precipitate that can easily be removed by sedimentation or filtration.

Various side reactions that take place during the reaction process are responsible for the enzyme inactivation (E) or inhibition (Eii) leading to a limited lifetime, but this form is not permanent since compound III (Eii) decomposes back to the resting state of peroxidase. Some peroxidases, like horseradish peroxidase (HRP), lead to a permanent inactivation state (P-670) when H₂O₂ is present in excess or when the end-product polymer adheres to its active site, causing its permanent inactivation by causing changes in its geometric configuration (Villalobos and Buchanan, 2002).

 

1.1.2    Classification of Peroxidase

Peroxidases, a class of enzymes in animals, plants and microorganisms, catalyze oxidoreduction reactions between hydrogen peroxide (H₂O₂) and various reductants. Peroxidases fall into two major super families according to their primary source: animal and plant peroxidases(Fleischmann etal., 2004).

 

1.1.2.1 Enzyme based peroxidase classification (EC)

Peroxidases can be grouped under the same enzyme classification number EC.1.11.1, donor: hydrogen peroxide oxidoreductase (Fleischmann etal., 2004). Currently, 15 different EC numbers have been ascribed to peroxidase: from EC 1.11.1.1 to EC 1.11.1.16 (EC 1.11.1.4 was removed) (Table 1). Two particular cases were also observed for EC 1.11.1.2 (NADPH peroxidase) and EC 1.11.1.3 (fatty acid peroxidase). Concerning EC 1.11.1.2, NADPH peroxidase activities have been observed in different studies (Conn etal., 1952; Hochman and Goldberg, 1991); however there is no known peroxidase sequence that has been assigned to this EC 1.11.1.2, probably due to the fact that none of the peroxidases known so far have a predominant NADPH peroxidase activity. Peroxidasins, peroxinectins, and other non-animal peroxidases, dyp type peroxidases, hybrid ascorbate cytochrome cperoxidase and other class II peroxidases do not possess an EC number. The two independent EC numbers (1.11.1.9 and 1.11.1.12) both correspond to gluthatione peroxidase and are based on the electron acceptor (hydrogen peroxide or lipid peroxide, respectively).

 

 

 

 

Table 1: The International Union of Biochemistry Classification of Peroxidases

Super Family	EC Number	Recommended Name	Abbreviation in Peroxibase	Molecular Weight (KDA)
Animal	EC 1.11.1.1	NADH peroxidase	NadPrx	50-55
	EC 1.11.1.2	NADPH peroxidase	No sequence available
	EC 1.11.1.3	Fatty acid peroxidase	No sequence available (aDox?)
	EC 1.13.11.11 (Previously EC 1.11.1.4)	Tryptophan 2,3 dioxygenase	Not considered as a peroxidase anylonger
Plant	EC 1.11.1.5	Cytochrome C peroxidase	CcP, DiHCcP	32-63
Catalase	EC 1.11.1.6	Catalase	Kat, CP	140-530
	EC 1.11.1.8	Iodide peroxidase	TPO
Animal	EC 1.11.1.9	Glutathione peroxidase	GPx	62-22 and 75-112
	EC 1.11.1.10	Chloride peroxidase	HalPrx, HalNPrx, HalVPrx
Plant	EC 1.11.1.11	l-ascorbate seperoxidase	APX	30-58
	EC 1.11.1.12	Phospholipid hydroperoxide Glutathione peroxidase	GPx
Plant	EC 1.11.1.13	Manganese peroxidase	MnP	43-49
Plant	EC 1.11.1.14	Lignin peroxidase	LiP	40-43
	EC1.11.1.16	Versatile peroxidase	VP
	EC 1.13.11.44	Linoleate diol synthase	LDS
Animal	EC 1.14.99.1	Prostaglandin endoperoxide Synthase	PGHS	115-140
	EC 1.6.3.1	NAD(P)H oxidase	DuOx
	EC 4.1.1.44	4 Carboxy muconolactone decarboxylase	AhpD, CMD, CMDn, HCMD, HCMDn, DCMD, DCMDn, AlkyPrx,AlkyPrxn

(Fleischman et al., 2004)

 

1.1.2.2 Haem Based Classification

In haem-based, sequences are organised according to the presence or abscence of haem. Sequences are divided between haem peroxidases and non-haem peroxidases (Figure 6). Genes encoding haem peroxidases can be found in almost all kingdoms of life. They are grouped into two major super families: one mainly found in bacteria, fungi and plants (Passardi etal., 2004) and the second mainly found in animals, fungi and bacteria (Daiyasu and Toh, 2000; Furtmuller etal., 2006). Members of the super family of plant/fungal/bacterial peroxidases (non-animal peroxidases) have been identified in the majority of the living organisms except in animals. Three independent classes can be distinguished: Class I, which includes ascorbate peroxidase (APx), cytochrome c peroxidase (CcP) and catalase peroxidase (CP); Class II, includes lignin peroxidases (LiP), manganese peroxidases (MnP), versatile peroxidase (VP) and Class III includes all plant peroxidases (Ruiz-Duenas etal., 2001).

The second super family described as “animal peroxidases” comprises a group of homologous proteins mainly found in animals and categorized as follows: myeloperoxidase (MPO); eosinophil peroxidase (EPO); lactoperoxidase (LPO); thyroid peroxidase (TPO); prostaglandin H synthase (PGHS); peroxidasin (Pxd) and peroxinectin (Pxt) as shown in (Figure 2). Homologous animal peroxidase sequences from the fungal kingdom can also be classified as PGHS.

Figure 2:Heam presence based peroxidase classification.(Passardi et al., 2004).

1.1.2.3 Non-Haem Containing Peroxidase

These peroxidases are mainly grouped in the following superfamilies: alkylhydroperoxidase D-like superfamily (AhpD, CMD, HCMD, DCMD, and AlkyPrx), NADH peroxidase (NadPrx), manganese catalases (MnCat) and two subfamilies of thiol peroxidases: glutathione peroxidases (GPx) and peroxiredoxins (1CysPrx, 2CysPrx, PrxII/PrxV/PrxGrx and PrxQ/BCP)(Passardi et al., 2004, Figure 2).Haem and non-haem peroxidases are found in all kingdoms; they may become key markers for the evolution of living organisms. Peroxi-Base represents a powerful tool for an efficient analysis and a better understanding of the evolution of protein superfamilies, catalytic domains and peroxidase activity in all kingdoms of life.

 

1.1.3    Structure of Peroxidase

Figure 3: Heam structure of horseradish peroxidase (Vietch, 2004)

Horseradish peroxidase C has two metal centers, one of iron haem group and two calcium atoms (Veitch, 2004) (Figure 3).The haem group has a structure with the iron atom held tightly in the middle of a porphyrin ring which is comprised of four pyrrole molecules. Iron has two open bonding sites, one above and one below the plane of the haem group. The second histidine residue in the distal side of the haem group, above the haem group, is vacant in the resting state (Vietch, 2004).  This site is open for hydrogen peroxide to attach during reduction-oxidation reactions. An oxygen atom will bind to this vacant site during activation.  The iron atom’s sixth octahedral position is considered the active site of the enzyme. During the enzyme catalysis, the binding of the hydrogen peroxide to the iron atom creates an octahedral configuration around the iron atom.  Other small molecules can also bind to the distal site, creating the same octahedral configuration (Vietch, 2004).

1. His170 forms coordinate bond with haem iron
2. Asp 247 carboxylate side-chain help to control imidazolate character of His170 ring.

III.   His170 Ala mutant undergoes haem degradation. When hydrogen peroxide is added and    compound I and compound II are not detected, imidazole can bind to haem  Iron in the artificially created cavity but full catalytic activity is not restored because the His170 imidazole complex does not maintain a five coordinate state (His42 also binds to Fe).

1. Aromatic substrates are oxidized at the exposed haem edge but do not bind to haem Iron

Figure 4: Carbohydrate components of HRPC (Veitch 2004).

 

Site of glycosylation is in loop regions of the structure, at Asn57, Asn13, Asn158, Asn186, Asn198, Asn214, Asn255 and Asn268.  The major glycan is shown here, there are also minor glycans of the form Manm GlcNA_c2. (m = 4 to 7) and (Xyl) xManm (Fuc)f GlcNAc2(m = 2, 4, 5, 6; f = 0 or 1; x = 0 or 1) (Figure 4).

Figure 5: Amino acid residues of HRPC (Veitch, 2004).

The haem group and haem iron atom are shown in red, the remaining residues in atom colours. His170, the proximal histidine residue, is coordinated to the haem iron whereas the corresponding distal coordination site above the plane of the haem is vacant (Veitch, 2004).

Amino acid residues

Arg38              Essential role in (i) The formation and stabilization of compound I,

(ii) Binding and stabilization of ligands and aromatic substrates (e.g. benzhydroxamic acid, ferulate etc.).

Phe41              Prevent substrate access to the ferryl oxygen of compound I.

His42               Essential role in (i) Compound I formation (accept proton from H₂O₂),

(ii) Binding and stabilization of ligands and aromatic substrates.

Asn70              Maintains basicity of His42 side-chain through Asn70-His42 couple (hydrogen)bond from Asn70 amide oxygen to His42 imidazole NH).

Pro139             Part of a structural motif, -Pro-X- Pro- (Pro139-Ala140-Pro141 in HRP C), which is conserved in plant peroxidases (Figure 5).

Figure 6: The 3 dimensional crystal structure of HRPC. (Brookhaven accession code 7H5A) (Veitch, 2004).

The haem group (coloured in red) is located between the distal and proximal domains which each contain one calcium atom (shown as blue spheres).α-Helical and β-sheet regions of the enzyme are shown in purple and yellow, respectively. The F^׳and F^״α-helices appear in the bottom right-hand quadrant of the molecule (Figure 6).

 

1.1.4    Mechanism of Action of Peroxidase

All peroxidases studied so far share much the same catalytic cycle that proceeds in three distinct and essentially irreversible steps (Dunford, 1991) and is often referred to as the “peroxidase ping-pong.” The resting ferric enzyme reacts with H₂O₂ in a two-electron process to generate the intermediate known as compound I. Compound I is discharged in two sequential single-electron reactions with reducing substrate yielding radical products, which are often highly reactive, and water. The first reduction step results in the formation of another enzyme intermediate, compound II. In the final step compound II is reduced back to ferric peroxidase (Figure 8). The peroxidase ping-pong provides an adequate description of the peroxidase reaction; however, continuing work has revealed some limitations of the basic model. The compound I reduction steps have been shown to consist of reversible substrate binding followed by substrate oxidation (Rodriguez-Lopez, et al., 2000). The formation and nature of compound I have been studied intensively. Both a neutral peroxidase-peroxide complex and a charged complex (known as compound 0) have been observed (Rodriguezet al., 2001), and variations have been identified in the electronic structures of the compound I of different peroxidases.

 

1.1.4.1 Mechanisms of oxidation of indole-3-acetic acid with peroxidase

One of the most interesting reactions of peroxidase (HRP-C) occurs with the plant hormone, indole-3-acetic acid (IAA) as shown in Figure 7. In contrast to most peroxidase–catalysed reactions, this takes place without hydrogen peroxide, hence the use of the term ‘indole acetic acid oxidase’ to describe this activity of HRP C in the older literature. More recent studies of the reaction at neutral pH indicate that it is not an oxidase mechanism that operates, but rather a peroxidase mechanism coupled to a very efficient branched-chain process in which organic peroxide is formed (Dunford, 1999). The reaction is initiated when a trace of the indole-3-acetate cation radical is produced. The major products of indole-3-acetic acid oxidation include indole-3-methanol, indole-3-aldehyde and 3-methylene-2-oxindole, the latter most probably as a result of the non-enzymatic conversion of indole-3-methylhydroperoxide. Conflicting theories have been proposed to explain the mechanism of reaction at lower pH(Dunford, 1999), in the formation of the ferrous enzyme, compound III and hydroperoxyl radicals must also be accounted for. The physiological significance of IAA metabolism by (HRP C) and other plant peroxidases is still an area of active debate. For example, studies on the expression of an anionic peroxidase in  transgenic tobacco plants indicate that while overproduction of the enzyme favours defensive strategies (such as resistance to disease, physical damage and insect attack), it has a negative impact on growth due to  increased IAA degradation activity (Lagrimini, 1996). Thus peroxidase expression in plant tissues at different stages of development must reflect a balance between the priorities of defense and growth.

Figure 7: A mechanism proposed for the formation of 3-methylene-2-oxindole from horseradiperoxidase (HRP C) and indole-3-acetic acid (Folkes et al., 2002).

R represents a cellular nucleophile (e.g. sulphydryl groups of enzymes or histone).

 

1.1.4.2 Mechanism of oxidation of small phenolic substrates (Ferulic acid) with peroxidase

Ferulic acid ((3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid. FA) is a phenolic cinnamic acid derivative that is abundant in nature and known to act as an in vivo substrate for peroxidases (Fry, 1986). FA enhances the rigidity and strength of plant cell walls by cross-linking with pentosans, arabinoxylans, and hemicelluloses, thereby making the cell walls less susceptible to enzymatic hydrolysis during germination. The compound is a dibasic acid that exhibits an extended resonance stabilization of the phenolate anion, hence slightly increasing its acidity relative to phenol. pKavalues of 4.6 and 9.4 have been reported (Kenttamaa et al., 1970)Peroxidases have been reported to be the FA cross-linking catalyst (Markwalder and Neukom, 1976). The level of FA and its derivatives seems to be positively correlated with protection of the plant against insects (Suga et al., 1993), fungal,viral andavian attacks. In plants, FA is thought to arise from the conversion of cinnamic acid and frequently it is esterified to hydroxyl groups of polysaccharides (Takahama and Oniki, 1996), flavonoids, hydroxycarboxylicacids and plant sterols. The initial step in the biosynthesis of lignin is the enzymatic dehydrogenation of monolignols to produce phenoxyl radicals. The radicals can link up to form dimers, trimers, and higher oligomers.

Laccases and plant peroxidases have been proposed to be the in vivo generators of the phenoxyl radicals. Peroxidase oxidation of compounds with a syringyl group can be enhanced by esters of 4-coumaric acid and fatty acid (Takahama and Oniki, 1996). For these reasons, it is of interest to study the interactions between the cell wall component of ferulic acid  and the well characterized horseradish peroxidase C. Peroxidases catalyze the oxidative coupling of phenolic compounds using H₂O₂ as the oxidizing agent as shown.  The reaction is a three-step cyclic reaction by which the enzyme is first oxidized by H₂O₂ and then reduced in two sequential one-electron transfer steps from reducing substrates, typically a small molecule phenol derivative (the charges of heme propionates are ignored in  scheme 1).

 

HRPC[(Fe(III))Porph^2-]⁺ + H₂O₂→ HRPC[(Fe(IV)=O)Porph^0-]⁰⁺ + H₂O …………Reaction(II)

Native state                                                   Compound I

HRPC[(Fe(IV)=O)Porph^0-]⁰⁺ + AH → HRPC[(Fe(IV)=O)Porph^2-]  + H⁺ +  A⁰ …….Reaction (III)

Compound II

HRPC[(Fe(IV)=O)Porph^2-] + H⁺  + AH  → HRPC[(Fe(III))Porph^2-]⁺  +  H₂O + A⁰….Reaction(IV)

 

SCHEME 1: Reaction II–IV

The oxidized phenolic radicals polymerize with the final product depending on the chemical character of the radical, the environment, and the peroxidase isoenzyme used (Frias et al., 1991).  The oxidation of native enzyme by H₂O₂ is well understood, and numerous experiments have confirmed the general catalytic mechanism for this step first proposed by (Poulos and Kraut, 1980). The oxidation of phenolic substrates (reactions III and IV) is less well understood, but a histidine(His42 in HRPC) and an arginine (Arg38 in HRPC) (Rodriguez-Lopez et al., 1997) have been shown to contribute significantly to enhance the rate of substrate oxidation.

 

1.1.4.3 Reactions of Peroxidases

Figure 8: Proposed mechanism for substrate oxidation in plant peroxidases (Veitch, 2004).

 

First, the active site arginine (Arg38 in HRPC) donates a hydrogen bond to the phenolic oxygen of the reducing substrate. This hydrogen bond will assist proton transfer from the phenolic oxygen to active site histidine (His42 in HRPC) through an active site water molecule held in position by the backbone oxygen of a conserved proline residue (Pro139 in HRPC). The electron is transferred to the haem group via the C-18 methyl-C-20 haem edge. Then compound II reduction is assisted by a similar proton transfer. The proton can be transferred to the ferryl oxygen through the active site water molecule situated equidistant between the distal histidine and the expected position of the ferryl oxygen of compound II, regenerating the resting state enzyme and a water molecule.

Figure 9: The proposed pathway to compound I formation for a typical heam peroxidase (Filizola and Gilda, 2000).

 

The postulated pathway to compound I formation from the inactive resting form of a typical heme peroxidase is shown schematically in Figure 9. As seen in this figure, peroxide is a requirement in the formation of compound I from the ferric resting form of the haem peroxidase. In the first step, peroxide binds to the ferric resting species (Figure 9a), forming a putative transient intermediate peroxide complex (Figure 9b). In this complex, peroxide is bound as a ligand to the haem iron. This species is thought to then undergo protein-assisted conversion to an oxywater complex (Figure 9c). It is the oxywater form (Figure 9c) that is thought to undergo facile O-O bond cleavage, leading directly to formation of compound I and water. Thus, the transformation of the peroxide complex to the oxywater complex is a key postulated step in compound I formation. The protein environment is thought to facilitate this key transformation process by aiding in both proton abstraction from the ligand peroxide oxygen atom (O₁) and proton addition to the distal peroxide oxygen (O₂) (Filizola and Gilda, 2000). Upon compound I formation, typical peroxidase substrates such as phenols, aromatic amines, and aromatic sulfonates are oxidized by two sequential one-electron oxidations. As a result, two radical products are formed, and the enzyme returns to its ferric resting state. The initial radical products are reactive transient species, and there are several possible fates of them. For example, the two radicals may dimerize, react with another substrate molecule, or attack another species causing co-oxidation. They may reduce molecular oxygen to superoxide or may be scavenged by molecular oxygen to form a peroxyl radical.

Figure 10: Alternative possible pathway to compound I formation (Filizola and Gilda, 2000).

 

At acid pH, compound I formation involves dynamic exchange of peroxide oxygen atoms as ligands for the haem iron (Figure 10). In the first step Arg38 acts as a proton donor to the O₂ peroxide ligand atom (a). Subsequently, the iron ligand changes from O₂ to O₁, permitting the formation of an incipient water molecule (b). In this mode, the hydrogen on O₁ can be abstracted by H₂O ₄₀₀, leading to formation of the oxywater complex and compound I (Figure 10b).

 

1.1.5Functions of Peroxidases

Peroxidase has more functions than a Swiss army knife, (Passardi et al., 2004).

 

1.1.5.1 Diverse Role of Class III Peroxidases   

In striking contrast to the first two peroxidase classes, assigning a function to a class III peroxidase is a rather complex task. Probably as a consequence of the large number of genes and the two possible catalytic cycles, class III plant peroxidases are involved in a broad range of physiological processes (Fig. 11). Plant peroxidases are involved in cell wall metabolism (Barcelo and Pomar, 2001; Passardi et al., 2004), wound healing (Bernards et al., 1999), and auxin catabolism (Gazaryan and Lagrimini, 1996) during their life cycle. They are also believed to be involved in the removal of H₂O₂, oxidation of toxic reductants, defense against pathogen or insect attack, as well as in symbiosis and in normal cell growth. In addition class III peroxidases can generate highly reactive ROS (Passardi et al., 2004) which can possess an intrinsic activity during different environmental responses and developmental processes, including the oxidative burst, the hypersensitive response (HR), or cell elongation (Bolwell et al., 2002; Bindschedler et al., 2006). Alternatively, ROS can also act as part of signal transduction pathways (Laloi et al., 2004) during specific mechanisms, including biotic and abiotic stress responses, allelopathic plant–plant interactions, cell division/elongation, and programmed cell death (Bethke and Jones, 2001). Plant peroxidases have often been suggested to be involved in the biosynthesis of complex cell wall macromolecules such as lignin and suberin, both of which are synthesized by plant for mechanical strength, defense, restoring damaged tissues, and water transport (Vidali, 2001). Plant peroxidases (PODs) oxidise phenolic domains of feruloylated polysaccharides and tyrosine residues of cell wall structural proteins such as hydroxyproline-rich glycoproteins to form more complex and larger molecules in the cell wall, thereby restricting cell expansion and pathogen invasion. In tobacco, a positive correlation was found between PODs activity and resistance to tobacco wildfire disease. The roles of PODs in defense are considered as follows:

1. Reinforcement of cell wall physical barriers comprising lignin, suberin, feruloylated polysaccharides and hydroxyproline-rich glycoproteins.
2. Enhancement of reactive oxygen species production as signal mediators and antimicrobial agents.

• Enhancement of phytoalexin production.

Generally, multiple PODs are induced by pathogen infection, suggesting that each POD is involved in a specific defense process (Hiraga et al., 2001;Cosio and Dunand,2009).

Figure 11: The diverse function and role of class III peroxidase (Cosioand Dunand,2009).

 

1.1.5.2 Degradation of Pesticides and Polychlorinated Biphenyls (PAHs)

Pesticides include a broad range of substances most commonly used to control insects, weeds, and fungi. Pesticide exposure is associated with chronic health problems or health symptoms such as respiratory problems, memory disorders, dermatologic conditions, cancer, depression, neurologic deficits, miscarriages and birth defects (McCauley et al., 2006). Peroxidases extracted from some fungal species have great potential to transform several pesticides into harmless form(s). Transformation of organophosphorus pesticides by white-rot fungi has been studied (Jauregui et al., 2003) and transformation of several organophosphorus pesticides by the chloroperoxidase from Caldariomyces fumago has been reported.

 

1.1.5.3 Functions of Peroxidase in Pharmacology and Fine Chemistry

Recently, peroxidases have been used as reagents for organic syntheses and biotransformations, as well as in coupled enzyme assays, chemiluminescent assays and immunoassays.

1. Oxidative decarboxylation of auxin (IAA), a plant hormone that affects many physiological processes by PODs (from tobacco and HRP). PODs induce IAA inactivation, thereby offering new potential for target cancer therapy. Studies reported that IAA is cytotoxic to human tumour cells in the presence of POD. The mechanism of toxicity involves 3-methylene-2-oxindole which is generated through IAA oxidation. Many other substituted indole-3- acetic acid derivatives have been tested for cytotoxicity in combination with HRP C in an attempt to place relationship between structure and activity on a predictive level. No simple correlation was found between levels of cytotoxicity of indole derivatives and their reactivity towards compound I; for example 5-fluoroindole- 3-acetic acid is more cytotoxic towards tumour cells than IAA but less effective as a reductant of compound I (Folkes et al., 2002). Other factors such as the pKa of the indolyl radical cation and rates of decarboxylation and radical fragmentation may also be significant.  One of the most cytotoxic indoles identified from in vitro screening is 6-chloroindole-3-acetic acid, a derivative with potential as a pro drug for targeted cancer therapies mediated by HRP C (Rossiter et al., 2002). The challenge now is to develop strategies to evaluate and implement this promising system in vivo. Indeed the combination of HRP C and indole-3-acetic acid or its derivatives offers several advantages for future antibody-, gene- or polymer-directed enzyme pro-drug therapies (Folkes and Wardman, 2001; Wardman, 2002). Among the favourable properties of HRP C are its good stability at 37^oC, high activity at neutral pH,   lack of toxicity and the ease with which it can be conjugated to antibodies and polymers. Furthermore, evidence available at present suggests that IAA does not show any adverse side-effects in humans. The fact that peroxide is not required as a co-substrate for the reaction with HRP C is also a significant advantage.
2. Some applications of HRP in small-scale organic synthesis include N- and O-dealkylation, oxidative coupling, selective hydroxylation and oxygen-transfer reactions.

• Peroxidase-catalysed oxidative coupling of methyl-(E)-sinapate with the syringyl lignin- odel compound, 1-(4-hydroxy-3,5-dimethoxyphenyl) ethanol yielded a novel spirocyclohexadienone together with a dimerization side-product.

1. Coupling of catharanthine and vindoline to yield α-3,4-anhydrovinblastine. This reaction, catalysed by HRP, offers potential interest as it is a semisynthetic step in the production of the anti-cancer drugs vinblastine and vincristine from Catharanthus roseus (Vidali, 2001;Veitch, 2004).
2. Peroxidases have also shown an action on tyrosine, both as free amino acid and in peptides or proteins. After one electron oxidation and subsequent deprotonation, dityrosines and higher oligomers are produced.
3. Ferulic acid and tyrosine are subject to peroxidase-mediated oligomerization. Such peroxidase-mediated hetero-coupling could provide an explanation for the occurrence of protein-carbohydrate complexes in plant cell walls and the incorporation of ferulic acid and other hydroxycinnamic derivatives into lignin and suberin tissues on a protein template. Recent studies have further explored the mechanism of hetero-adduct formation of GYG (Gly-Tyr-Gly) and FA (Ahn et al., 2002).

• Reactive oxygen species (ROS) generated through abiotic and biotic stresses trigger programmed cell death (PCD) in mammalian cells, yeast and plants (Delannoy et al.,2005). In plants and yeast the PCD is induced by Bax proteins that cause organelle dysfunction by their localisation onto the outer mitochondrial membranes and formation of ion channels. Several enzymes have been reported to suppress Bax-induced cell death such as peroxidase, ascorbate peroxidase, peroxidase with glutathione transferase and phospholipid hydroperoxid glutathione peroxidase (Chen et al., 2004; De Gara, 2004).
• Many studies have suggested an association of plant peroxidases with production and scavenging of hydrogen peroxide, porphyrin metabolism, senescence and organogenesis, indicating that PODs have diverse functions (Hiraga et , 2001). Based on previous published works (El Agha et al., 2009; Osman et al., 2008) for the exploitation and valorization of crude POD from cheap vegetable sources.

 

1.1.5.4    The Use of Peroxidase for Wastewater Treatment

Although the use of enzyme in the waste water treatment was first proposed in the 1930s only as late as in the 1970s the concept of environmental biocatalysts that is, application of enzymes to destroy target pollutant was established. Enzyme may transform pollutant to diminish their toxicity, to increase water solubility and its subsequent removal from the industrial waste stream. Peroxidase was shown to be able to remove a variety of phenols and aromatic amines from an aqueous solution (Klibanov and Morris, 1981) and to decolorize phenolic and amines industrial effluents. It was shown that phenols are effectively removed by treatment with horseradish peroxidase in the presence of a coagulant. However, peroxidase quickly becomes inactivated during the reaction, and the coagulant prevents peroxidase inactivation and reduces the amount of peroxidase required for phenol removal. Arseguel and Baboultne (1994) studied the removal of phenol using peroxidase in the presence of a mineral and showed that the mineral could prolong the catalytic action because of the adsorption of the reaction products. Enzyme immobilization is excellent due to its high storage stability and better control of the catalytic process (Tatsumi et al., 1994).

 

1.1.5.5 The Use of Peroxidase in Textile Industry

Most synthetic industrial dyes are complex aromatic compounds with an azo bond connected to various aromatic structures. Some, however, are polymeric structures containing metals. It is   estimated that there are over 10 000 commercially available dyes and pigments of industrial use, representing an annual consumption of around 7 x 10⁵ tonnes worldwide (Akhtar et al., 2005). However, about 10-15% of the synthetic dyes produced are discharged into industrial effluents (Spadaro et al., 1992), causing environmental problems. Then, dye contamination of water bodies is a great problem in many countries. Removal of dyes can be carried out by means of oxidative enzymes. Peroxidases, a versatile group of enzymes that catalyze the oxidation of a large number of aromatic structures through a reaction with hydrogen peroxide, being applied in the chemical, environmental, pharmaceutical and biotechnological industries (Spadaro et al., 1992).

 

1.1.5.6 The Use of Peroxidase in the Dairy Industry

Hydrogen peroxide has been use in the dairy industry as an effective bactericidal and bacteriostate agent, although its mechanism of action is unclear. The bacterial reduction by H₂O₂ depends on the initial quality of the milk (i.e. the bacterial count) (Nambudripad et al., 1949). However, H₂O₂ used as a preservation in the dairy industry to preserve the milk against microbial spoilage can lead to the destruction of the physical properties,chemical composition and original nutritional value.These H₂O₂ used in dairy industry either to preserve or to kill bacterial can be destroyed easily and quickly and completely through the use of peroxidase, after  enzymatic treatment, the breakdown products, water and oxygen are normally undetected in milk and  no toxic residue remains once H₂O₂ has been broken down.

1.1.5.7 Application in Analysis and Diagnostic Kits

Due to the horseradish peroxidase (HRP) ability to yield chromogenic products at low concentrations and its relatively good stability, it is well-suited for the preparation of enzyme conjugated antibodies and application in diagnostic kits. In analytical application(s), the enzyme must be present in saturating amounts to make sure that the H₂O₂ produced in the test is stoichiometrically converted into a colored substance (Krell, 1991). Various peroxidase isoenzymes have been purified from roots and hairy-roots cultures of turnip (Brassica napus) (Agostini et al., 2002). They developed a diagnostic test kit for determination of uric acid. The assay was based on the following reactions:

Uric acid +O₂ + 2H₂O Uricase → Aallantoin + H₂O₂ + CO₂ …………………………Reaction (V)

H₂O₂ + 4-aminophenazone + phenol peroxidase → p-(Benzoquinone) monoiminephenazone

Horseradish peroxidase (HRP) is the most commonly used enzyme for practical analytical applications. However, peroxidases from other sources appear to be a good alternative as substitutes for HRP. Peroxidase isoenzyme from turnip hairy roots could be used as a reagent for clinicaldiagnosis, as part of a kit where H₂O₂ is generated (Krell, 1991).

 

1.1.5.8 Enzyme Linked Immunosorbent Assay (ELISA)

Horseradish peroxidise (HRP) is probably the most common enzyme used as a reporter (enzyme-labelledantibody) in enzyme immunoassays. HRP-containing ELISA kits, have been appliedin food control, diagnostic microbiology and as disposable amperometric immuno-sensors (Green et al., 2004). Thewidespread application of ELISAs for analytical purposes is due to the extremely high selectivityand affinity of antibody molecules to their corresponding antigens. An ELISA tests in whichperoxidase is used for labelling an antibody, have been developed for screening monoclonalantibodies against mycotoxins (Kawamura et al., 1989). Usefulness of ELISA using peroxidase as conjugated protein indiagnosis of human prion disease has been reported (Yuki et al., 2011). By using maize,wheat, rye, barley as by-products, a useful ELISA has been developed in order to detect T-2 toxinhaving detection limit of 50 ng/g (Sibanda et al, 2000).

 

1.1.5.9Applications in Paper Pulp Industry

Biopulping is a process where extracellular enzymes (hydrolytic and oxidative) produced by a white-rot fungus remain adsorbed on the wood chips thus leading to degradation of lignin (Arana etal., 2002).The pulping by-products cause serious environmental problem due to its high pollution load. In order to degrade these by-products, many potential fungal and bacterial strains can be applied. Two bacterial strainsCitrobacter freundii (FJ581026) and Citrobacter sp. (FJ581023) were applied in axenic and mixed condition for degradation of black liquor (pulping by product). The optimum activity of lignin degrading enzyme was noted at 96 h and characterized as manganese peroxidase by SDS-PAGE analysis (Chandra and Abhishek, 2011). Ligninolytic haem peroxidases were able to break down the main linkage types in lignin due to their high redox-potential and specialized catalytic mechanisms (Martinez 2007; Hammel andCullen, 2008). White-rot fungi attacked lignin and simultaneously degraded wood components to carbon dioxide and water (Arana et al., 2002). Direct use of microorganisms for breaking down of lignocellulosic materials had many drawbacks, including degradation of cellulose fibres (Jimenez and Martinez, 1997) and long reaction times, extending to several days (Katagiri et al., 1995).

 

1.1.5.10Organic and Polymer Synthesis

HRP has been used to polymerize phenolic and aromatic amine compounds (Oguchi et al.,1999). HRP catalysed the oxidative coupling of methyl-sinapate with a syringyl lignin model compound 1-(4-hydroxy-3, 5-dimethoxyphenyl) ethanol in the presence of H₂O₂. Currently, polyaniline is synthesized by oxidizing monomer aniline under strongly acidic conditions and low temperature using ammonium persulfate as the initiator of radical polymerization (Rannou et al., 1998). In nature, peroxidases have good stability at low pH, making them a good alternative for polymerizing aniline under acidic conditions. Using an anionic peroxidase purified from the African oil palm tree, an enzymatic synthesis of the polyelectrolyte complex of polyaniline and also sulfonated polystyrene has been developed (Sakharov et al., 2003). In the presence of H₂O₂, peroxidase catalyzes the oxidation of phenols that eventually give rise to high molecular weight polymers (Nicell and Wright, 1997). This characteristic could be used as an attractive alternative to the conventional formaldehyde method used for the production of lignin-containing phenolic resins.

 

1.1.5.11Deodorization of Swine Manure

The HRP could be used as an enzymatic source in the deodorization of swine slurry (Govere et al., 2005). Odorant compounds such as phenols, indoles, volatile fatty acids, ammonia, hydrogensulphide and mercaptans are either initially present in manure or result from anaerobictransformation of animal wastes (Hobbs et al., 1995; Zahn et al., 1997). Elevated odour level inconfinement buildings can reduce livestock growth rates, thereby increasing the outbreaks ofinfections and adversely affecting farm workers (Hardwick, 1985). Treatments, such as dietarymanagement, intense aeration or zone treatment and the application of manure additives havebeen used to decrease or eliminate odorous compounds (Hobbs et al., 1995). HRP has been proven an effective alternative for deodorization ofmanures. Minced horseradish with calcium peroxide reduced the concentration of phenol by 70%and for VFAs by 45%. A 100% reduction in the concentration of phenolic odorants withoutreoccurrence within 72 hrs was achieved by using HRP (Govere et al., 2005).

 

1.1.5.12Peroxidase Biosensors

Peroxidase has great potential in the field that comprising electrochemical biosensors. Peroxidase- based electrodes have had widespread use in analytical systems for determination of H₂O₂ and organic hydroperoxides (Jia et al., 2002). When co-immobilized with a H₂O₂ producing enzyme, they may be exploited for determination of glucose, alcohols, glutamate and, choline (Ruzgas et al., 1996).The importance of determination of H₂O₂ lies in the fact that H₂O₂ plays an important role in clinical, chemical, biological, environmental and many other fields (Tripathi et al., 2006;Shi et al., 2007).A novel third generation biosensor for H₂O₂ was constructed by cross-linking HRP onto an electrode modified with multiwall carbon nanotubes (MWNTs) (Xu et al., 2011). Compared with other analytical techniques, electrochemical enzyme biosensors had the advantage of high selectivity of the biological recognition elements and high sensitivity of the electrochemical transduction process. In this respect, a novel immobilization platform was developed by synergistically using ZnO crystals and nano-sized gold particles as HRP-loading material (Zhang et al., 2009).

 

1.1.5.13Fungal Peroxidases for Biofuel Production

Indeed, the world’s strongest economies are deeply committed to the development of technologies aimed at the use of renewable sources of energy. Within these agenda, the substitution of liquid fuel gasoline by renewable ethanol is of foremost importance. Biomass hydrolysis, i.e. the depolymerization of the biomass polysaccharides to fermentable sugars to produce ethanol and other biofuels, must be performed via environmentally friendly and economically feasible technologies (Lynd et al., 2005). The enzyme based application is advantageous over chemical treatments due to its higher conversion efficiency, the absence of substrate loss due to chemical modifications and the use of more moderate and non-corrosive physical-chemical operating conditions, such as lower reaction temperatures, ‘neutral pH’ and the use of biodegradable and nontoxic reagents (Lynd et al., 2005).

Bio-ethanol and other biofuels produced from lignocellulosic biomass represent a renewable, more carbon-balanced alternative to both fossil fuels and corn-derived or sugar-cane-derived bio-ethanol. Unfortunately the presence of lignin in plant cell walls impedes the breakdown of cell wall polysaccharides to simple sugars and subsequent conversion of these sugars to useable fuel. To achieve an optimal biological conversion of lignocellulosic biomass to biofuel, lignin must be physically removed from plant tissue before saccharification (Weng etal., 2008). One of the most common fates of lignin in nature is to be metabolized by lignin peroxidases, manganese peroxidases and closely-related enzymes of white rot basidiomycetes (Hammel and Cullen, 2008). Peroxidases have potential in deigning enzymatic biofuel cells which are attractive for a number of special applications, such as disposable implantable power suppliers for medical sensor-transmitters and drug delivery; they offer practical advantages of using abundant organic raw materials as biofuels for clean and sustainable energy production (Weng etal., 2008).

 

1.1.5.14Pharmaceutical Industries

Haem peroxidases have the potential to be widely used as catalysts in fine chemical preparations. This is because they are enzymes capable of performing a wide variety of oxidation reactions, ranging from radical coupling reactions, to oxygen-atom insertion into substrates, to several types of halogenation processes (Aehle, 2007). This delocalization of the radical on the phenol nucleus accounts for the product composition, since the radical coupling can occur through an ortho-ortho processing, giving rise to an O-biphenyl adduct, or an ortho-para process, forming the so called Pummerer’s ketone, a pharmacophoric synthon (Valenti et al., 2006). The problems connected with the high cost and low stability of peroxidases, which limits their potential applications in processes of industrial interest, could be possibly overcome, at least in part, by the use of haem-peptide complexes as small-size peroxidase analogs,using micro-peroxidases, the haem-peptide complexes obtained from proteolytic degradation of cytochrome c, as starting point for the preparation of complexes with improved activity and controlled substrate selectivity (Casella et al.,2000; Lombardi et al., 2001).

 

1.1.5.15      Application as Bleaching Detergents

Several patent applications have appeared mainly generated by research groups within Novo-Nordisk A/S and their considerable activity in this area. One of the first patent in which this novel lead has been disclosed is that by Kirk and Farrel (1987). The claim is made that an enzyme exhibiting peroxidase activity is capable of exerting a bleaching effect on fabrics. The main advantage would be that by using the detergent additives of the invention amounts of hydrogen peroxide or its precursors can be reduced and yet provide a satisfactory bleaching effect.  A more specific application, is that of the inhibition of dye transfer during washing or rinsing of fabrics by addition of enzyme exhibiting peroxidase activity to the wash liquor. Since the peroxidase will oxidize or bleach the bleeding dye during the wash, the peroxidase will inhibit the transfer of a textile dye from a dyed fabric to another fabric when these fabrics are washed and/or rinsed together in wash liquor. This problem is most noticeable when white or light coloured fabrics are washed together with fabric with a more intense colour from which the dye is leached during washing. Peroxidases has been reported for their use in removing excess dye from newly dyed or printed textiles by using wash liquor containing oxidase or peroxidase. The advantage of this treatment is that it bleaches any dye leaching from the material so it prevents redeposition of dye (backstaining). It reduces time, energy and water, produces less polluted wash water and improves dye fastness (Kirk and Farrel, 1987).

 

1.2       Substrates

Peroxidases are found mainly as haemoproteins and use hydrogen peroxide as the oxidizing substrate. In reactions of peroxidase, a variety of electron donors are used including aromatic amines, phenols and enedioles like ascorbic acid to detect peroxidase activity in plant extract. For a reaction to occur with peroxidase, two substrates must be present i.e hydrogen peroxide and any other from the aromatic amines, phenols, enedioles like ascorbic acid etc. The choices of this second substrate vary depending on the cell or tissue the enzyme is found.   In this study a dye like o-dianisidine was used as electron donor to detect peroxidase because it gives stable colored oxidized product and shows high sensitivity for the reaction (Ila and Mahanta, 2012).

1.2.1    Hydrogen Peroxide

Hydrogen peroxide is best known for its use as an oxidising agent. Its strong oxidising potential allows it to oxidise a large number of organic and inorganic compounds (Ila and Mahanta, 2012).

Peroxidase

It can however act as a reducing agent for strong oxidants. When it decomposes, it forms water and releases oxygen, which makes it an attractive “environmental friendly” product. It is “a clean oxidant”.  This ability to act both as reductant and oxidant allows hydrogen peroxide to react in a wide range of applications. With water as its only by-product, hydrogen peroxide is ideal for chemical reactions or syntheses where by-products would be undesirable (Reaction VI).

H₂O₂ + ZH₂                                                2H₂0 + Z    …………………………………….Reaction(VI)

H₂O₂ is a common end product of oxidative metabolism, and being a strong oxidizing agent, could prove toxic if allowed to accumulate. Thus, peroxidases serve to rid plant cells of excess H₂O₂ under normal and stress conditions (Ila and Mahanta, 2012).

Its oxidizing action is used for:

• Bleaching paper pulp.
• Bleaching textile and plant fibres.
• Manufacturing chemical compounds and preparing other oxidants.
• Destroying pollutants and toxic substances.
• Metal surface treatment, minerallurgy and uranium hydrometallurgy.

It also features outstanding disinfectant and antiseptic properties which are exploited in many applications.

 

1.2.2    Other substrates

The various substrates that can react with peroxidase and their respective products are shown in Table 2. Oxygen atom is transferred to an acceptor molecule, which for example is the organic molecule o-dianisidine. This reaction is facilitated by the enzyme peroxidase, which is found in many plant tissues. Peroxidase is more in horseradish rather than in turnips. Hydrogen peroxide is the primary substrate for peroxidase and any of the phenolic compounds as shown in Table 2.

4H – R – O – H + 4H – O – O – H      R – O – O –  R+8H2O ……….Reaction VII

 

 

 

 

Table 2: Various substrates that can react with peroxidase and their respective products.

Substrates	Products
Pyrogallol	Purpurogallin
Guaiacol	Tetraguaiacol quinone
Benzidine	o-Quinonediamide
Catechol	o-Quinone
Hydroquinone	Quinhydroine
Tyrosine	Yellow solution
o-Cresol	Milky precipate
m – Cresol	Flesh-coloured solution
p-Cresol	Green-solution
o-Dianisidine	Vivid purplish red

 

The general reaction is as shown below;

1.2.2.1 O-dianisidine

O-dianisidine dihydrochloride also known as 3,3-dimethoxybenzidine dihydrochloride is a carcinogenic compound used in the assay of peroxidases. It is an aromatic amine that is initially a col­orless crystal but turns violet upon standing at room temperature (HSDB, 2009). It is practically insoluble in water, but is soluble in al­cohol, benzene, ether, chloroform, acetone, and probably most other organic solvents. It is stable at normal temperatures and pressures (Lebedeva et al., 1977).

Figure 12: Reactions of peroxidase with O-dianisidine and H₂O₂

 

 

1.2.2.2 Physical and chemical properties of o-dianisidine.

Molecular Formula	C14H16N2O2
Molecular weight (molar mass)	244.3g/mol
Melting point	268oC
Boiling point	393oC
Flash point	403oF
Density	1.178g/cm3

 

1.3       Factors that Affect Peroxidase Activity

1.3.1    pH

This is a measure of hydrogen ion activity of a solution and is defined as the negative logarithm of the hydrogen ion concentration. The rate of a chemical reaction or the enzyme activity is greatly influenced by the structure of the enzyme. The peroxidase activity was low above and below the optimum pH. The pH at which an enzyme catalyzes a reaction at the maximum rate is called the optimum pH. Studies have shown several the effect of pH on the activities of peroxidase from different sources.Allium sativum peroxidase showed optimum activity at pH 6.0, a rapid decrease in activity was found on either the basic or acidic side of this optimum pH (Haq et al., 2012). Similar optimum pH (6.0) was observed for peroxidase from broccoli (Thongsook and Barrett, 2005) and Armoracia lapathifolia leaves (Saitou et al., 1991), to lettuce stems (Yihong et al., 2012), Armoracia rusticana (Lavery et al., 2010) and Eruca vesicaria sbsp. Sativa (Nadarogluet al., 2013). The pH of peroxidase purified from delicious apple was 5.0 to 6.0, royal delicious apple 7.0 (Muhammed et al., 2011) and turnip, 5.0 (Agostini et al., 2002). The enzyme was stable over a narrow range of pH from 5.0 to 7.0 after 24 hrs incubation at 4ºC; the residual activity at pH 9.0 was 22%.

This is the summary of the effect on pH and on a combination of these factors: The binding of the enzyme to substrate, the catalytic activity of the enzyme, the ionization of the substrate, and the variation of protein structure.

1.3.2    Temperature

Temperature is one of the critical factor affecting enzyme-catalyzed reactions, like other chemical reactions, the rate of an enzyme-catalyzed reaction increases with modest increase in temperature. This is true only over a strictly limited range of temperature. When the temperature of a reaction is raised, there is sufficient energy to overcome the energy barrier and so cause an increase in the number of collision between the enzyme involved and its substrate. These result in an increase in the rate of the reaction to reach its maximum activity. Beyond optimum temperature, every further increase in temperature introduces vibrational energy that weakens the three-dimensional structure of the enzyme. Once the hydrogen bonds and hydrophobic bonds holding the native structure together are broken or disrupted, the enzyme is denatured and the reaction stop. The temperature range over which an enzyme is stable and catalytically active depends on the temperature of the cell in which the enzyme is found (McEldoon and Dordick, 1996).As temperature increases, the rate of reaction also increases, as is observed in many chemical reactions. However, the stability of the protein also decreases due to thermal degradation. Holding the enzyme at a high enough temperature for a long period of time may cook the enzyme or inactivate it. It was observed that the maximum temperature for peroxidase activity was between 30^oC and 70^oC in most vegetables and fruits that have been studied (Majed and Mohammad, 2005). Inactivation temperature of peroxidase has been reported  to be 95^oC in soybean seed coat peroxidase,  81.5^oC in horseradish peroxidase C and that of Caprinus cinereus peroxidase (a class II POD from the  fungus Caprinus cinerus with similar activity) is 65^oC (McEldoon and Dordick, 1996). That of litch POD was 90^o C for 10 minutes, and 100^oC for 1 minute.

All of the POD fractions are present in plant tissues as a combination of various isoenzymes with different thermal stability (Eze et al., 2010). The differences in heat resistance of the isoenzymes vary considerably with the vegetable source and origin. The peroxidase from Eruca vesicaria sbsp. Sativa was stable at 40°C and lost 18 and 19% of its activity after 50 and 60 min at 40°C, respectively. This purified peroxidase has high thermal stability. So it indicates that it is an excellent enzyme for the pharmaceutical, food and detergent industries. (Nadagrolu et al., 2013). Most plant peroxidases show optimum activity in the temperature range of 30 to 45°C (Ila and Mahanta, 2012). The rate of thermal inactivation of three quality-related enzymes in white yam was measured over the temperature range 50-80°C. It was observed that the time and temperature of the heating process affected the rate of inactivation of the enzyme (Eze et al., 2010b).

It is important to note that at higher temperatures, response to temperature would appear to follow a biphasic denaturation pattern suggesting that inactivation occurs by more than one mechanism each with its own temperature dependence (Eze et al., 2010b).

 

1.4       Inhibition and Inhibitors of Peroxidase

Many substances alter the activity of an enzyme by combining with it in a way that influences the binding of the substrate or its turnover number. Substances that reduce an enzyme‘s activity in this way are known as inhibitors. Many inhibitors are substances that structurally resemble their enzyme’s substrate but either does not react or reacts very slowly compared to the substrate.

Inhibition of enzymes decreases yield of products and finally the affectivity of the process. There exists two prepositions concerning the mechanism of enzyme inactivation. The first one hypothesizes intermediates (radicals), a formation that reacts with active centre of enzyme(Chang et al., 1999). Following the second hypothesis micro particles adsorb enzyme (Masuda et al., 2001). However, the intrinsic mechanism of inactivation of absorbed enzyme is not understood.

1.4.1    Inhibitor of Peroxidases/ Peroxidase Suppressor

Horseradish peroxidases are inhibited by thiol type inhibitor: mercaptoethanol (MCE) and mercaptoacetic acid (MCA) Mercaptoethanol (MCE) is a more potent inhibitor than mercaptoacetic acid using 4-aminoantipyrine as a substrate. Other inhibitors arep-aminobenzoic acid, sodium azide (NaN₃), cyanide, hydroxylamine, sulfide, sulfite, vanadate and a number of divalent anions of Cd, Co, Cu, Fe, Mn, Ni, Pb (Nicelland Wright, 1997).

1.5       Gongronema latifolium Leaves

1.5.1    Gongronema latifolium

Gongronema latifolium is known as ‘utazi’ in the southeastern and ‘arokeke’ in the south-western part of Nigeria. Also Gongronema latifolium is called “madumaro” by Yoruba ethnic group in Nigeria It is a perennial edible plant with soft and pliable stem. It is a tropical rainforest plant which belongs to the family of Aslepiadaceae(Ugochukwu and Babady, 2002; Ugochukwu et al., 2003). It is a climber with tuberous base found in deciduous forest from Guinea Bissau and western Cameroons. Various parts of these plants, particularly the stems and leaves are used as chewing sticks or liquor and in places such as Sierra Leone they are also used as a decoction or cold infusion of pounded stem is used for colic and intestinal symptoms usually associated with worm (Deighton, 1957).The liquor, usually obtained after the plant is sliced and boiled with lime juice or infused in water over three days is usually taken as a purge for colic and stomach pains as well as to treat symptoms connected with worm infections (Okafor, 1981).  In Ghana the boiled fruit are used as laxative. In Eastern State of  Nigeria, the leave are used to prepare food for mother that have recently put to bed, where it is believed to stimulate appetite, reduce post-partum contraction and enhance the return of the menstrual cycle (Morebise et al., 2002).

The plant is also widely used in folk medicine as a spice and vegetable (Morebise et al., 2002) for maintaining healthy blood glucose levels (Okafor, 1981). Antibacterial activity of the leaf extract has also been reported (Nwinyi et al., 2008). The use of medicinal plants in curing diseases is as old as man (Grabley and Thiericke, 1999; Abinu et al., 2007). These plants which are found in our environment enjoy wide acceptability by the population and serve as cheaper alternatives to orthodox medicine (Sofowora, 1993; Akah and Nwabie, 1994). Gongronema latifolium is one of such medicinal plants whose therapeutic application has a folkloric background. The plant enjoys widespread reputation as a remedy for inflammation, bacteria, ulcer, malaria, diabetes and analgesic. Hence a scientific verification of its uses would be important in establishing a pharmacological basis for some of the claimed ethno medicinal uses (Sofowora, 1993).

1.5.2    Physiological Properties of Gongronema latifolium Plant

Gongronema latifolium, most common name is amaranth globe. The parts commonly used are the leaves, stem and root. The origin of the plant is traced to Nigeria in West Africa. It is a rainforest plant which has been traditionally used in the South Eastern part of Nigeria over the ages for the management of diseases such as diabetes and high blood pressure(Okafor, 1981). G latifolium is a woody tropical plant. It has bitter taste and the ideal soil for growing it is red late rite soil. It is a slender climber, often 3–4 m long, but able to climb to the canopy of high trees, with woody base and fleshy roots, containing latex.  The leaves are opposite, simple, softly hairy; petiole up to 4 cm long; blade ovate, 5–14 cm × 3–10 cm, base cordate, apex acuminate, margins entire. Inflorescence is cymose is composed of 2–3 primary branches divided dichotomously, each division ending in a 10–14-flowered umbel. The flowers are  bisexual, small, regular, 5-merous, yellow-green; pedicel is 1 cm long; sepals elliptical-oblong, 2 mm × 1 mm; corolla tubular, with campanulate tube up to 4 mm long, lobes elliptical-oblong, is 2 mm long, spreading; corona lobes as long as stamens; stamens with deltoid to ovate anther appendages, connivent around the stout, roundish style apex. The fruit is a pair of leathery, pendent follicles, each one cylindrical, 10–15 cm× 4–8 mm, densely brown-grey hairy. Gongronema is a small genus comprising 5 species in Africa, much resembling Dregea (Ugochukwu et al., 2003).

Figure 13: The leaves of Gongronema latifolium

 

 

Figure 14:  Farm land showing Gongronema latifolium climbing on sticks

 

1.5.3    Chemical Composition of Gongronema latifoliumLeaves

The chemical composition of Gongronema latifolium leaves has been determined using standard methods. The aqueous and methanol, crude protein, lipid extracts, ash, crude fibre and nitrogen free extractives obtained are: 27.2%, 6.07%, 11.6%, 10.8% and 44.3% dry matter respectively (Afolabi, 2007). Their potassium, sodium, calcium, phosphorus and cobalt contents are 332, 110, 115, 125 and 116 mg/kg respectively. The dominant essential amino acids are leucine, valine and phenylalanine. Aspartic acid, glutamic acid and glycine are 13.8%, 11.9% and 10.3% respectively of total amino acid content. Saturated and unsaturated fatty acids are 50.2% and 39.4% of the oil respectively. Palmitic acid makes up 36% of the total fatty acid (Afolabi, 2007), vitamin A, C, and E contents are 21.29, 27.40, 3.19 u/100g respectively. Riboflavin, thiamine, niacin comprise 0.96, 0.18 and 0.81% respectively (Atangwho et al., 2009).

 

1.5.4    Microbial Studies on the Gongronema latifoliumLeaves

6. latifolium extracts were tested against thirteen pathogenic bacterial isolates. The extracts show no activity against E. faecalis, Y. enterolytica, E. aerogenes, B. cereus and E. agglomerans. The methanol extracts were active against S. enteritidis, S. cholerasius ser typhimurium and P. aeruginosa with minimum inhibitory concentration (MIC) 1 mg; zone of growth inhibition 7, 6.5 and 7 mm respectively.The aqueous extracts show activity against E. coli (MIC 5 mg) and P. aeruginosa (MIC 1 mg) while methanol extracts are active against P. aeruginosa and L. monocytogenes. G. latifolium has potential food and antibacterial uses (Afolabi, 2007).

 

1.5.5    Phytochemical Compositions of Gongronema latifolium

Phytochemical analysis of G. latifolium showed that it contains alkaloids, acidic compounds, flavonoids, saponins, tannins, resins, steroids and essential oils as shown in Table 5. These classes of compounds have some curative effect on micro-organisms induced disease. The natural products are good in several ways, flavonoids are found to be antimicrobial. It is used as a seasoner in food, which may be bitter or sweet or astringent (Uhegbu et al., 2011).

 

 

 

 

Table 3: Phytochemical and anti-nutrient content (%) of Gongronema latifoliumleaves.

Alkaloid	9.40
Flavonoid	0.042
Saponin	2.70
Steroid	4×10-3
Hydrogen cyanide	5× 10-4
Tannin	6.10
Starch	ND
Anthocyanin	ND

(Uhegbu etal., 2011)                                       ND= Not Detected

 

1.5.6    Uses of Gongronema latifolium

1. The antioxidant and antitussive properties of Gongronema latifolium used locally by Nigerian poultry farmers for the treatment of fowl cough was investigated (Essien et al., 2007). It was stated that the leaf extract significantly reduced the mortality rate of the broilers by 25% within 3 weeks of treatment and by 40% in 6 weeks of administration, when the broilers were 13 weeks old. The reduction in mortality coincided with reductions in the microbial loads in the trachea of the sick 7-week old broilers.
2. The anti-oxidative properties of Gongronema latifolium are being utilized in management of diabetes (Ugochukwu et al., 2003) Traditionally, the leaf is believed to stimulate appetite, reduce post-partum contraction,  enhance the return of the menstrual cycle and used in controlling weight gain in lactating women(Nwanjo et al., 2006).

• The spice has been used historically to improve the anti-oxidation and anti -malaria activity of food. Gongronema latifolium is one of the plants used as spice for flavouring, seasoning, and imparting aroma to food.

1. Gongronema latifoliumhas been investigated to be nutritionally high in iron, zinc,vitamin, protein and amino acid, thus could complement the inadequacies of these substance in feed (Agbo et al., 2006).
2. Also the bitter principles when extracted may have potential in beer brewing (Adenuga et al., 2010).

 

1.6       Enzyme Thermostability and Thermodynamics

The use of plants as bioreactors is not a new concept. Within the last fifteen years, studies have established that with the knowledge of biotechnology and genetic engineering, plants are indeed a low-cost source to produce stable molecules: plants are harnessed to produce antibodies, biodegradable plastics, recombinant proteins, carbohydrates, and fatty acids. A major goal of plant-based bioreactor technology is the production of stable enzymes to produce industrially useful products.Special characteristics of microbial and plant enzymes include their capability and appreciable activity under abnormal conditions, mainly of temperature and pH. Hence, certain plant enzymes are categorized as thermophilic, acidophilic or alkalophilic. Plants with systems of thermostable enzymes that can function at higher than normal reaction temperatures would decrease the possibility of microbial contamination in large scale industrial reactions of prolonged durations (Wang et al., 2012). The quality of thermostability in enzymes promotes the breakdown and digestion of raw materials; also the higher reaction temperature enhances the penetration of enzymes (Zhang and Wu, 2011). The complete saccharification and hydrolysis of polysaccharides containing agricultural residues requires a longer reaction time, which is often associated with the contamination risks over a period of time. Therefore, the hydrolytic enzymes are well sought after, being active at higher temperatures as well as retaining stability over a prolonged period of processing at a range of temperatures. The high temperature enzymes also help in enhancing the mass-transfer and reduction of the substrate viscosity (Berka et al., 2011) during the progress of hydrolysis of substrates or raw materials in industrial processes. Thermophilic xylanase are considered to be of commercial interest in many industries particularly in the mashing process of brewing. The thermostable plant xerophytic isoforms of laccase enzyme are considered to be useful for their applications in textile, dyeing, pulping and bioremediation (Chirumamilla et al., 2001).

Several plant enzymes have a significant place in the list of microbial enzymes, which have established their applications in bio-industries. Lipases have been widely studied for their properties and utilization in many industries (Reddivariet al., 2002). Pectinases have established their role in the fruit and juice industries (Sunnotel and Nigam, 2002). Certain enzymes are specifically required in pharmaceutical industry for diagnostic kits and analytical assays (Zhouet al., 1995).

Bornscheuer et al. (2012) have currently mentioned that in all the research and developments so far in the field of biocatalysis, the researchers have contributed in three waves of outcomes. These innovations have played an important role in the establishment of current commercially successful level of bio-industries. As a result recent bioprocess-technology is capable of meeting future challenges and the requirements of conventional and modern industries, for example Trincone (2013) has reviewed the options for unique enzymatic preparation of glycosides. Earlier enzymatic process were performed within the limitations of an enzyme, whereas currently with the knowledge of modern techniques, the enzyme can be engineered to be a suitable biocatalyst to meet the process requirement. Riva (2013) has identified the scope of a long-term research in biocatalysis, since there are underlying problems in the shift from classical processes to bio-based processes for commercial market.

Biotechnology is utilizing a wide range of enzymes synthesized on a commercial scale employing purposely screened plants and microorganisms. Selected plants and microorganisms have been characterized, purposely designed and optimized to produce a high-quality enzyme preparation on large scales for industrial applications. Different industries require enzymes for different purposes; hence microbial enzymes have been studied for their special characteristics applicable in various bio-processes. Recent molecular biology techniques have allowed to tailor a specific plant, to produce not only the high yields of an enzyme, but also enzyme with desired special characteristics such as thermostability, tolerance at high temperature and its stability in acidic or alkaline environment, and retaining the enzyme activity under severe reaction conditions such as in presence of other metals and compounds.The thermostable enzymes isolated fromplants have just started providing conversions under conditions that are appropriate for industrial applications. The conditions required by these thermostable enzymes which bring about specific reactions not possible by chemical catalysts are still mild and environmentally benign, as compared to the temperatures and pressures required for chemical conversions. Thus, with the availability of thermostable enzymes a number of new applications in the future are likely. Although, believed to provide tremendous economic benefits, production of the enzymes to the level required by the industries has remained a challenge.

Thermostability is the ability of enzyme to resist against thermal unfolding in the absence of substrate, while thermophilicity is the capability of enzymes to work at elevated temperatures in the presence of substrate (Sarath Babu et al., 2004, Bhati et al., 2006). Thermal denaturation may occur in two steps as shown below:

…………………Reaction (IX)

 

Where, E_act is native enzyme, E_inact is unfolded enzyme that could be reversibly refolded upon cooling and X is the denatured enzyme formed after prolonged exposure to heat and therefore cannot be recovered on cooling. In recent years, interest in thermostable enzymes has increased dramatically as resistance to thermal inactivation has become a desirable property of the enzymes used in many industrialapplications. Thermostable enzymes are generally defined as those with an optimum temperature above that of the maximum growth of an organism or with exceptional stability above 50ºC over an extended period of time (Singh et al., 2000). One of the ways to identify enzymes, which are thermally stable, is to exploit natural sources. Temperature produces opposed effects on enzyme activity and stability and it is therefore a key variable in any biocatalytic process (Wasserman, 1984). Biocatalyst stability i.e the capacity to retain activity through time is undoubtedly the limiting factor in most bioprocesses, biocatalyst stabilization being the central issue of biotechnology (Illanes, 1999). Biocatalyst thermostability allow a higher operation temperature, which is clearly advantageous because of higher reactivity, higher process yield, lower viscosity and fewer contamination problem (Mozhaev, 1993). Enzyme thermal inactivation is the consequence of weakening the intermolecular forces responsible for the preservation of its 3D-structure, leading to a reduction in its catalytic capacity (Misset, 1993). Inactivation may involve covalent or non-covalent bond disruption with subsequent molecular aggregation or improper folding (Bommarius and Broering, 2005). Knowledge on enzyme inactivation kinetics under process condition is an absolute requirement to properly evaluate enzyme performance (Illanes et al., 2008). Various thermostability parameters, such as half-life of the enzyme preparation (T_1/2), the activation energy of thermal denaturation (E_aD), enthalpy of activation of the thermal denaturation (ΔH_D), entropy of activation of thermal denaturation (ΔS_D) and the Gibbs free energy of activation of thermal denaturation (ΔG_D) were used to evaluate the thermostability of peroxidase extracted from Gongronema latifolium.

 

 

 

1.7       Aim and Objectives of the Study

1.7.1    Aim of the Study

This investigation was carried out onproduction,partial purification from Gongronema latifolium leaves that might be thermostable for industrial uses

1.7.2    Specific Objectives of the Study

This study was designed to achieve the following objectives.

• Extraction of peroxidase from Gongronema latifolium
• Protein determination of the peroxidase in Gongronema latifolium
• Assay for peroxidase activity using O-dianisidine.
• Purification of the enzyme using ammonium sulphate (NH₄)₂SO₄ precipitation and gel filtration chromatography.
• Characterization of the partially purified Gongronema latifoliumperoxidase based on pH, temperature and substrate concentrations.
• Thermal-inactivation and regeneration of the peroxidase in Gongronema latifolium

 

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