PH AND THERMAL STABILITIES OF PEROXIDASE ISOLATED FROM RIPENING TOMATO FRUITS (SONALUM LYCOPERSICON)

CHAPTER ONE

INTRODUCTION

The super-family of peroxidases from plants, fungi and bacteria are haem proteins that

catalyse the oxidation of various electron donor substrates (phenols,  aromatic amines)  at  the expense of hydrogen peroxide (Welinder, 1992). Peroxidases contain iron (III)  protoporphyrin IX (ferriprotoprophyrinIX), as the prosthetic group. They have a molecular weight ranging from

30 to 150kD and may be divided into mammalian, microbial and plant peroxidases (Welinder,

1992). The haem peroxidases are ubiquitous in nature with diverse physiological functions, and are classified  into three groups on the basis of their amino acid sequences (Welinder,  1976). 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  (Kim  and  Lee,  2005).  Compound  I is subsequently reduced by reactions 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).  Class I  includes  intracellular  peroxidases,  cytochrome  c peroxidase,  ascorbate  peroxidase,  and  gene-duplicated  bacterial  catalase-peroxidase.  Class  II contains secretory fungal enzymes, such as manganese peroxidase and lignin peroxidase. Class III consists of secretory plant peroxidases (Welinder, 1992). There is increasing interest in these classes  of  peroxidase,  not  only to  establish  their  physiological  roles,  but  also  for  possible industrial  and  analytical  applications  (Segel,  1993).  Microbial  peroxidases  are  involved  in lignification of cell walls (Blee et al., 2003) and in the metabolism of the plant hormone indole-

3-acetic  acid  (IAA)  (Veitch,  2004).  Studies  have  suggested  that  peroxidases  play a role  in lignification,  suberization,  cross-linking  of cell wall structural  protein,  auxin  catabolism  and self–defense   against   radical-mediated   peroxidation   of  unsaturated   lipids,  pathogens   and senescence (Hiraga et al., 2001) and help with salt tolerance and oxidative stress (Smith et al.,

2003). In rubber trees, peroxidase was found in newly excised bark strip, possibly in response to wounding, i.e. excision (Bolwell et al., 2002). In sunflower leaves, it may also be involved in leaf senescence (Clemente, 1998).

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 (Duran and Esposito, 2000). In this treatment, phenolic compounds are polymerized  in the  presence of hydrogen peroxide through a radical oxidation-reduction  mechanism (Nazari  et  al., 2005).   Peroxidase  is widely used commercially as secondary antibody for research  and medical diagnosis (Colonna et al.,

2002), as indicators for reactive oxygen species formed during food processing (Thongsook and Barret, 2005) and as catalysts for de-lignification of paper pulp (Farrell et al., 2007). The other areas where peroxidase could have an immediate use and economic impact are soil remediation (Reetesh  and  Kunwar,  2011)  and  the  synthesis  of  polyelectrolyte  complexes  (Mackey  and Killard, 2007). Recently, the combination of peroxidase and IAA has been introduced as a novel cancer therapy (Egorov et al., 2004). Although peroxidases are found from various sources such as fungi, plants are the traditional  sources for  commercial  production of peroxidases  having attracted comparatively more attention than other sources for commercial purposes because of advantages such as superior stability and increased sensitivity (Wei, 2003).

1.1       Peroxidases

Peroxidases (EC1.11.1.7) are oxido-reductases that catalyze the reduction of peroxides, such  as  hydrogenperoxide  (H2O2)  and  the  oxidation  of  a  variety  of  organic  and  inorganic compounds (Welinder, 1976). The catalytic cycle involves distinct  intermediate enzyme forms (Krell  et  al.,  1992).  The   r e a c t i o n   of  peroxidase-catalyzed  reaction  is  depicted  in  the following general equation:

AH2 + H2O2 → A+ 2H2O. ( Chattopadhyay and Mazumdar, 2000) Specifically, peroxidase activity involves donating electrons that bind to other substrates such  as  ferricyanides  and  ascorbate,  in  order  to  break  them  into  harmless  components. Peroxidases degrade H2O2, a naturally occurring by-product of oxygen metabolism in the body. As a result, these substances  are converted  to water and oxygen  (Suha et al., 2013). By the

early1900s, as yet unknown enzymes at work in human body were labeled as ‘catalases’ while the simultaneous observation that plants and animals utilized polyphenols to degrade H2O2 led to the term ‘peroxidases’(Marzouki et al., 2005 ). Peroxidases play important roles in protection of plant leaves from salt-induced oxidative damage (Duarte-Vazquez et al., 2005). The well-known

and best studied peroxidase is horseradish peroxidase (HRP) (Welinder, 1976) but other plants like Solanum lycopersicon sourced peroxidases have attracted comparative attention because of their availability (Wei, 2003).

Peroxidases  are  versatile  biocatalysts  with an ever  increasing  number  of  applications (Veitch, 2004). Plants peroxidases contain two-calcium ions (Ca2+), which are essential for their structural and thermal stabilities as well as in vitro activation during  analysis (Sticher et al.,

1981). Peroxidase activity has been shown to arise from the presence of quite a large numbers of isoenzymes varying in substrate specificity, heat stability, molecular weight, isoelectric point and immunological  properties  (Lee and Pennesi,  2008; Vidziunaite  et al., 2003;  Wakamatsu  and Takahama,  2009).  However,  unlike  many other  plant  isoenzymes,  the  iso-electric  points (pI values) for isoperoxidases traverse a wider range of pH values,  generally from pH 4 to 10 as typified by Lycopersicon species (McLellan & Robinson, 2009; Moulding et al., 1988).

Peroxidase is an enzyme found in many plant-based foods. The enzyme is of concerns to food processors because of its high thermostability and its involvement in the oxidation of many organic compounds, leading to deterioration in flavor, color, and nutritional quality (Manu and Prasada-Rao, 2009). These qualitative changes have been found to occur particularly in canned and frozen fruits and vegetables during storage  (Annele, 2012). Peroxidase is also used as an index of the adequacy of fruit and vegetable blanching due to its presence in most plant tissues, its high thermal stability, and the simplicity of its measurement (Anthon and Barrett, 2002). Heat treatment  is commonly used  to inactivate an active enzyme.  However,  it is well-known  that Peroxidase can recover its activity after heat treatment (Schwimmer, 2001). Many studies have revealed that residual or reactivated peroxidase can cause significant deterioration in the quality of various high-temperature-short-time  (HTST) processed foods (Adams, 2012) Reactivation of the  enzyme  is suggested  to  be  a  complex  process  and  is  influenced  by several  factors.  In horseradish  peroxidase  (HRP),  reactivation  has  been  reported  to  take  place  after   partial inactivation at 70, 90, or 110°C (Tamura and Morita, 2011). One factor affecting its reactivation is the time taken to reach the desired treatment temperature. If this time is short, reactivation occurs more easily (Rodrigo et al, 2000). This fact poses a problem in HTST treatment of acid and low-acid vegetables that are subsequently frozen or canned  (Lu and Whitaker, 2004). The ability of peroxidase to regain activity after being denatured by heat varies with the species of vegetable and may even differ between isoenzymes of the same species (Halpin et al, 1999).

1.1.1 Physiological functions of peroxidase

Most  reactions  catalyzed  by  peroxidase  especially  horseradish  peroxidase  can  be expressed by the following equation, in which AH2 and AH represent a reducing substrate and its radical  product  respectively.  Typical  reducing  substrates  include  aromatic  phenols,  phenolic acids, indoles, amines and sulfonates (Villalobos and Buchanan, 2002).

H2O2 + 2AH2 + POD → 2H2O + 2A…………………………………………………..Equation 1

Figure 1: Catalytic reaction 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 (AH) 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 (AH) and the return of the enzyme to its resting state, completing the catalytic reaction (Figure 1). The two free radicals (AH) 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), leads to a permanent  inactivation  state when H2O2  is present in excess or when the end-product polymer adheres to its active site, causing its permanent inactivation due to changes in its geometric configuration (Villalobos and Buchanan, 2002).

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