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

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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.
TABLE OF CONTENTS
Page
Title Page … .. .. .. .. .. .. .. .. .. .. i
Certification … .. .. .. .. .. .. .. .. .. .. ii
Dedication … .. .. .. .. .. .. .. .. .. .. iii
Acknowledgement … .. .. .. .. .. .. .. .. .. iv
Abstract … .. .. .. .. .. .. .. .. .. .. v
Table of Contents … .. .. .. .. .. .. .. .. .. vi
List of Figures … .. .. .. .. .. .. .. .. .. .. xi
List of Tables … .. .. .. .. .. .. .. .. .. .. xiii
List of Abbreviations … .. .. .. .. .. .. .. .. .. xiv

CHAPTER ONE: INTRODUCTION
1.1 Peroxidase … .. .. .. .. .. .. .. .. 2
1.1.1 Functional roles of peroxidase … .. .. .. .. .. .. 3
1.1.2 Classification of peroxidase … .. .. .. .. .. .. 4
1.1.2.1 Enzyme-based peroxidase classification (EC) … .. .. .. .. 4
1.1.2.2 Haem-based classification … .. .. .. .. .. .. 6
1.1.2.3 Non-haem containing peroxidase … .. .. .. .. .. .. 7
1.1.3 Structure of peroxidase … .. .. .. .. .. .. .. 7
1.1.4 Mechanism of action of peroxidase … .. .. .. .. .. 10
1.1.4.1 Mechanisms of oxidation of indole-3-acetic acid with peroxidase … .. 10
1.1.4.2 Mechanism of oxidation of small phenolic substrates (Ferulic acid) with peroxidase 11
1.1.4.3 Reactions of peroxidases … .. .. .. .. .. .. 13
1.1.5 Functions of peroxidases … .. .. .. .. .. .. 15
1.1.5.1 Diverse role of class III peroxidases … .. .. .. .. .. .. 15
1.1.5.2 Degradation of pesticides and polychlorinated biphenyls (PAHs) … .. 17
1.1.5.3 Functions of peroxidase in pharmacology and fine chemistry … .. .. 17
1.1.5.4 The use of peroxidase for wastewater treatment … .. .. .. 19
1.1.5.5 The use of peroxidase in textile industry … .. .. .. .. 20
1.1.5.6 The use of peroxidase in the dairy industry … .. .. .. .. 20
1.1.5.7 Application in analysis and diagnostic kits … .. .. .. .. 21

1.1.5.8 Enzyme-linked immunosorbent assay (ELISA) … .. .. .. 21
1.1.5.9 Applications in paper pulp industry … .. .. .. .. .. 21
1.1.5.10 Organic and polymer synthesis … .. .. .. .. .. .. 22
1.1.5.11 Deodorization of swine manure … .. .. .. .. .. .. 22
1.1.5.12 Peroxidase biosensors … .. .. .. .. .. .. .. 23
1.1.5.13 Fungal peroxidases for biofuel production … .. .. .. .. 23
1.1.5.14 Pharmaceutical industries … .. .. .. .. .. .. 24
1.1.5.15 Application as bleaching detergents … .. .. .. .. .. 25
1.2 Substrates … .. .. .. .. .. .. .. .. 25
1.2.1 Hydrogen peroxide … .. .. .. .. .. .. .. 26
1.2.2 Other substrates … .. .. .. .. .. .. .. .. 26
1.2.2.1 O-dianisidine … .. .. .. .. .. .. .. .. 27
1.2.2.2 Physical and chemical properties of o-dianisidine … .. .. .. 28
1.3 Factors that affect peroxidase activity … .. .. .. .. .. 28
1.3.1 pH … .. .. .. .. .. .. .. .. .. 28
1.3.2 Temperature … .. .. .. .. .. .. .. .. 28
1.4 Inhibition and inhibitors of peroxidase … .. .. .. .. .. 29
1.4.1 Inhibitor of peroxidases / peroxidase suppressor … .. .. .. .. 30
1.5 Gongronema latifolium leaf … .. .. .. .. .. .. 30
1.5.1 Gongronema latifolium … .. .. .. .. .. .. .. 30
1.5.2 Physiological properties of Gongronema latifolium plant … .. .. 31
1.5.3 Chemical composition of Gongronema latifolium leaves … .. .. 33
1.5.4 Microbial studies on the Gongronema latifolium leaves … .. .. 33
1.5.5 Phytochemical compositions of Gongronema latifolium … .. .. 33
1.5.6 Uses of Gongronema latifolium … .. .. .. .. .. .. 34
1.6 Enzyme thermostability and thermodynamics … .. .. .. 35
1.7 Aim and Objectives of the study … .. .. .. .. .. .. 38
1.7.1 Aim of the study … .. .. .. .. .. .. .. .. 38
1.7.2 Specific objectives of the study … .. .. .. .. .. .. 38

CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials … .. .. .. .. .. .. .. .. 39
2.1.1 Equipment … .. .. .. .. .. .. .. .. .. 39
2.1.2 Chemicals and reagents … .. .. .. .. .. .. .. 39
2.2 Methods … .. .. .. .. .. .. .. .. .. 39
2.2.1 Preparation of buffer solution … .. .. .. .. .. .. 39
2.2.1.1 Sodium phosphate buffer [0.05M; pH 6.0] … .. .. .. .. 39
2.2.1.2 Acetate buffer (stock solution) … .. .. .. .. .. .. 40
2.2.1.3 Tris-HCl buffer (stock solution) … .. .. .. .. .. .. 40
2.2.2 Determination of protein … .. .. .. .. .. .. .. 40
2.2.2.1 Preparation of reagent for protein standard curve … .. .. .. 40
2.2.2.2 Measurement of protein content … .. .. .. .. .. .. 40
2.2.3 Extraction of enzyme (peroxidase) … .. .. .. .. .. 41
2.2.3.1 Preparation of enzyme extract … .. .. .. .. .. .. 41
2.2.4 Preparation of substrate solution … .. .. .. .. .. .. 41
2.2.5 Peroxidase assay using o-dianisidine as substrate … .. .. .. 41
2.2.6 Purification of peroxidase from Gongronema latifolium leaves … .. .. 42
2.2.6.1.1 Ammonium sulphate precipitation profile … .. .. .. .. 42
2.2.6.1.2 Ammonium sulphate precipitation … .. .. .. .. .. 42
2.2.6.2 Gel Filtration chromatography … .. .. .. .. .. 42
2.2.6.2.1 Preparation of sephadex G-200 gel … .. .. .. .. .. 42
2.2.6.2.2 Selection of column and packing of gel … .. .. .. .. .. 43
2.2.6.2.3 Introduction and collection of enzyme … .. .. .. .. .. 43
2.2.7 Studies on the purified enzyme … .. .. .. .. .. .. 43
2.2.7.1 Characterization of enzyme … .. .. .. .. .. .. 43
2.2.7.1.1 Effect of pH on peroxidase activity … .. .. .. .. .. 43
2.2.7.1.2 Effect of temperature on peroxidase activity … .. .. .. .. 44
2.2.7.2 Kinetic study on the partially purified enzyme … .. .. .. .. 44
2.2.7.2.1 Effects of H2O2 on peroxidase activity … .. .. .. .. .. 44
2.2.7.2.2 Effects of different concentrations of o-dianisidine on peroxidase activity … 44
2.2.7.3 Estimation of kinetic parameters … .. .. .. .. .. 44

2.2.7.4 Determination of protein concentration and peroxidase activity … .. .. 45
2.2.8 Thermal inactivation and regeneration studies … .. .. .. .. 46
2.2.8.1 Determination of percentage residual activity … .. .. .. .. 46
2.2.8.2 Determination of thermal inactivation rate constant K (min-1) of the enzyme … 47
2.2.8.3 Determination of the half-life (t1/2) of the enzyme … .. .. .. 47
2.2.8.4 Determination of the D-value and Z-value … .. .. .. .. 47
2.2.8.5 Determination of activation energy of inactivation Ea … .. .. .. 47
2.2.8.6 Thermodynamics parameters … .. .. .. .. .. .. 48

CHAPTER THREE: RESULTS
3.1 Studies on crude enzyme … .. .. .. .. .. .. 49
3.1.1 Protein concentration of the crude enzyme … .. .. .. .. 49
3.1.2 Peroxidase activity of the crude enzyme … .. .. .. .. .. 49
3.2 Purification of the crude enzyme … .. .. .. .. .. .. 49
3.2.1 Ammonium sulphate precipitation profile of Gongronema latifolium peroxidase 49
3.2.2 Gel elution profile of partially purified Gongronema latifolium peroxidase … 49
3.3 Purification Table of Gongronema latifolium peroxidase … .. .. 52
3.4 Characterization of Gongronema latifolium peroxidase … .. .. .. 54
3.4.1 Effect of pH on Peroxidase Activity … .. .. .. .. .. 54
3.4.2 Effect of Temperature on Peroxidase Activity … .. .. .. .. 54
3.4.3 Effect of substrate concentration on peroxidase activity … .. .. 57
3.4.3.1 Effect hydrogen peroxide concentration on peroxidase activity … .. .. 57
3.4.3.2 Effect o-dianisidine concentration on peroxidase activity … .. .. 57
3.5 Determination of kinetic parameters of Gongronema latifolium peroxidase … 57
3.6 Characterization table of Gongronema latifolium peroxidase … .. .. 64
3.7 Thermal inactivation and regeneration studies of Gongronema latifolium peroxidase 65
3.7.1 Percentage residual activities of the enzyme … .. .. .. .. 65
3.7.2 Calculation of inactivation rate constants (K) of the enzyme … .. .. 65
3.7.3 Calculation of Half-life (t1/2) of the enzyme … .. .. .. .. 70
3.7.4 Calculation of D-Value and Z-Value … .. .. .. .. .. 70
3.7.5 Calculation of Activation Energy Ea of the enzyme … .. .. .. 70

3.7.6 Calculation of the thermodynamic parameters of the enzyme … .. .. 70
3.7.7 Percentage Regeneration Studies … .. .. .. .. .. .. 74

CHAPTER FOUR: DISCUSSION AND CONCLUSION
4.1 Discussion … .. .. .. .. .. .. .. .. 77
4.2 Conclusion … .. .. .. .. .. .. .. .. 85
4.3 Suggestions for further studies … .. .. .. .. .. .. 85
References … .. .. .. .. .. .. .. .. .. .. 86
Appendices … .. .. .. .. .. .. .. .. .. .. 101

LIST OF FIGURES
Figure 1: Catalytic cycle of peroxidase … .. .. .. .. .. .. 3
Figure 2: Heam presence based peroxidase classification … .. .. .. .. 6
Figure 3: Heam structure of horseradish peroxidase … .. .. .. .. .. 7
Figure 4: Carbohydrate components of HRPC … .. .. .. .. .. 8
Figure 5: Amino acid residues of HRPC … .. .. .. .. .. .. 8
Figure 6: The 3 dimensional crystal structure of HRPC … .. .. .. .. 9
Figure 7: A mechanism proposed for the formation of 3-methylene-2-oxindole from
horseradishperoxidase (HRP C) and indole-3-acetic acid … .. .. .. 11
Figure 8: Proposed mechanism for substrate oxidation in plant peroxidases … .. 13
Figure 9: The proposed pathway to compound I formation for a typical heam peroxidase … 14
Figure 10: Alternative possible pathway to compound I formation … .. .. .. 15
Figure 11: The diverse function and role of class III peroxidase … .. .. .. 17
Figure 12: Reactions of peroxidase with o-dianisidine and H2O2 … .. .. .. 27
Figure 13: The leaves of Gongronema latifolium … .. .. .. .. .. 32
Figure 14: Farm land showing Gongronema latifolium climbing on sticks … .. .. 32
Figure 15: Ammonium sulphate precipitation profile of peroxidase extracted from G. latifolium. 50
Figure 16: Gel elution profile of Gongronema latifolium peroxidase … .. .. 51
Figure 17: Effect of pH on peroxidase activity … .. .. .. .. .. 55
Figure 18: Effect of temperature on enzyme activity … .. .. .. .. 56
Figure 19: Effect of different concentrations of hydrogen peroxide on peroxidase activity
with time. .. .. .. .. .. .. .. .. .. .. 58
Figure 20: Michealis-Menten’s plot of hydrogen peroxide (H2O2) … .. .. .. 59
Figure 21: Effect of different concentrations of o-dianisidine on peroxidase activity with time 60
Figure 22: Michealis-Menten’s plot of o-dianisidine … .. .. .. .. .. 61
Figure 23: Lineweaver-Burk plot 1/v against 1/[H2O2] … .. .. .. .. 62
Figure 24: Lineweaver-Burk plot of o-dianisidine … .. .. .. .. .. 63
Figure 25: Percentage residual activity of Gongronema latifolium peroxidase for peak A … 66
Figure 26: Percentage residual activity of Gongronema latifolium peroxidase for peak B … 67
Figure 27: Determination of inactivation rate constants (K) of Gongronema latifolium
peroxidasefor peak A … .. .. .. .. .. .. .. 68
Figure 28: Determination of inactivation rate constants (K) of Gongronema latifolium
peroxidasefor peak B … .. .. .. .. .. .. .. 69
Figure 29: Plot of Log D against Temperature (˚C) to determine Z-Value for peakA … 71
Figure 30: Plot of Log D against Temperature (˚C) to determine Z-Value for peak B … 71
Figure 31: Arrhenius plot for thermal inactivation of Gongronema latifolium peroxidase
forpeak A … .. .. .. .. .. .. .. .. .. 72
Figure 32: Arrhenius plot for thermal inactivation of Gongronema latifolium peroxidase
for peakB … .. .. .. .. .. .. .. .. .. 72
Figure 33: Regeneration of Gongronema latifolium for peak A … .. .. .. 75
Figure 34: Regeneration of Gongronema latifolium for peak B … .. .. .. 76

LIST OF TABLES
Table 1: The International Union of Biochemistry classification of peroxidases … .. 5
Table 2: Various substrates that can react with peroxidase and their respective products … 27
Table 3: Phytochemical and anti-nutrient content (%) of Gongronema latifolium … .. 34
Table 4: Purification of Gongronema latifolium peroxidase … .. .. .. .. 53
Table 5: Characterization for Gongronema latifolium peroxidase … .. .. .. 64
Table 6: Thermo-inactivation parameters for Gongronema latifolium peroxidase for peak A… 73
Table 7: Thermo-inactivation parameters for Gongronema latifolium peroxidase for peak B… 73

LIST OF ABBREVIATIONS
POD Peroxidase.
H2O2 Hydrogen peroxide.
HRP Horseradish peroxidase.
H2O Water.
FeSO4 Iron II Sulphate.
BSA Bovine Serum Albumin.
mM Millimolar.
(NH4)2SO4 Ammonium sulphate.
KH2PO4 Di hydrogen potassium phosphate.
Tris-HCl Tris Hydrochloride (Amino hydroxyl methyl propane diol).
O-dia O-dianisidine.
OD Optical density.
KM Michaelis-Menten constant.
Vmax Maximum velocity.
IAA Indole acetic acid

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).

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