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 (Km) and maximum velocity (Vmax) 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 (Ea(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?)

Plant	EC 1.13.11.11   (Previously EC 1.11.1.4) EC 1.11.1.5	Tryptophan 2,3 dioxygenase           Cytochrome C peroxidase	Not considered as a   peroxidase anylonger       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
Plant	EC 1.11.1.12       EC 1.11.1.13	Phospholipid hydroperoxide Glutathione peroxidase Manganese peroxidase	GPx       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       EC 1.6.3.1	Prostaglandin endoperoxide   Synthase   NAD(P)H oxidase	PGHS       DuOx	115-140
	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.

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

1.1.2.3 Non-Haem Containing Peroxidase
gure 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).

I.      His170 forms coordinate bond with haem iron

II.    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).

IV.   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 GlcNAc2. (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).

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.

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