CHAPTER 6 ENZYME KINETICS AND THERMAL INACTIVATION OF POLYPHENOL OXIDASE

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1 CHAPTER 6 ENZYME KINETICS AND THERMAL INACTIVATION OF POLYPHENOL OXIDASE OVERVIEW OF CHAPTER Here we report the substrate specificity and enzyme kinetics of Polyphenol oxidase enzyme of A. paeoniifolius. The Km and Vmax values of PPO were measured by evaluation of the Lineweaver Burk (1/Vo versus 1/[S] values) plots. Thermal inactivation and thermodynamic properties of enzyme were checked for inactivation of polyphenol oxidase enzyme so that the application of enzyme is minimized in product development. 85

2 ENZYME KINETICS AND THERMAL INACTIVATION OF POLYPHENOL OXIDASE 6.1 INTRODUCTION The function of enzyme technology is to achieve the adequacy of industrial requirements for making it better than any other alternative process. One of the significant parameter among enzyme technology is thermal deactivation of enzyme under process condition, which is supposed to be a substantial factor in numerous biotechnological processes. Prompt inactivation may delay effectiveness of the process in spite of having good catalytic capability of the enzyme. Therefore enzyme deactivation kinetics is necessary for improved understanding of relations between structure and function of the enzymes to improve the viability of biotechnological process. Additionally, enzyme kinetics and thermodynamic parameters values are also helpful in analyzing the stability of proteins. Thermodynamic data of an enzyme catalysed reactions are important in the prediction of the amount of reaction as well as position of reactions process. The capability of enzyme is measured by catalysis in biochemical reactions while the stability of an enzyme is judged by its residual activity. Thermodynamic stability is defined by the enzyme s free energy of stabilization and its half-life (t 1/2 ) at distinct temperatures. Stability of PPO enzymes in altered conditions is essential to ensure the economic and technical viability for an industrial process. Thermodynamics acts as a major tool for understanding the deactivation process. This chapter utilises a thermodynamic approach (deactivation kinetics: Δ H #, Δ S #, Ea and Δ G # ) at different temperatures to understand the behaviour of Polyphenol oxidase enzymes. Present chapter aim towards better understanding of PPO enzyme kinetics, stability of the enzymes at higher temperatures ( C) and thermodynamic parameters which will be beneficial for thermal inactivation of enzyme for utilization in an industrial process. 86

3 6.2 MATERIAL AND METHODS Substrate specificity and Kinetics A. paeoniifolius PPO enzyme substrate specificity was determined by utilizing 50 mm concentration of various phenolic compounds as substrate solutions (viz. catechol, gallic acid, L tyrosine, DL tyrosine, pyrogallol, chlorogenic acid and resorcinol). For determination of Michaelis constant (Km) and maximum velocity (Vmax), PPO activities were measured using catechol as substrate at various concentrations ranging from 5 mm to 125 mm. The reaction velocity [V] was determined by monitoring change in absorbance per min. Km and Vmax values of the enzyme were calculated from the slope and intercept of the straight line plot of 1/V vs. 1/S( Lineweaver and Burk plot) Thermal stability of PPO enzyme Thermal stability of enzyme was determined where buffered enzyme mixture was incubated at the desired temperature C for 5 to 40 min in water bath, then rapidly cooled over ice bath so as to reach 35 C following which the substrate (catechol) was added and residual enzyme activity found. The residual enzyme activity is a ratio expressing relative enzyme activity (%) to the original unheated enzyme activity. The first order inactivation constant, (k) was determined from the slope of first order enzyme inactivation equation: log (% residual activity) = - (k/2.303) t [1] Half-life (t 1/2 ) value of inactivation is given by the expression: [2] Results of the thermal experiments were processed into kinetic parameter values, decimal reduction time (D) and its dependence on temperature expressed by Z. The decimal reduction time was calculated according to Stumbo (1973)[187] as: D = 2.303/k [3] The Z parameter was derived from log D values at different treatment time versus temperature. The Z value indicates how many degrees the temperature increase is required for decimal reduction time to be 10 fold higher or lower. 87

4 6.2.3 Thermodynamic analysis Arrhenius law is usually used to describe the temperature dependence of k-values, and it is algebraically given by ln (k)=ln(a) (Ea/R)*(1/T) [4] The activation energy (Ea) was estimated by slope of the straight line obtained using Arrhenius plot of the logarithm of the rate constant (log k) versus the inverse of absolute temperature. Where R is the universal gas constant (8.314 J mol -1 K -1 ), A is the Arrhenius constant, Ea is the activation energy (energy required for the inactivation to occur) and T is the absolute temperature in Kelvin. Obtained values of the Activation energy (Ea), the activation enthalpy (Δ H # ) for each temperature was calculated was by Δ H # = Ea-RT [5] The activation energy (Ea) was estimated by slope of the straight line obtained using Arrhenius plot of the logarithm of the rate constant (log k) versus the inverse of absolute temperature. The free energy of inactivation ( G # ) can be determined according to the expression: Δ G # = -R*T*ln (k* h P / K B *T) [6] Activation entropy (Δ S # ) calculated by Δ S # = ΔH # - G # /ΔT [7] Where K B is the Boltzmann constant (1.38 x J/K), h P is the Planck constant (6.626 x J.s), and T is the absolute temperature, k (s- 1 ) the inactivation rate constant of each temperature. 88

5 6.3 RESULTS AND DISCUSSION Substrate specificity The enzyme affinity varied depending on the substrate used. PPO specificities were examined in the presence of different substrates (Figure 6.1). PPO showed substrate preference for diphenols. Enzyme preferred catechol (considered as 100 %,) over chlorogenic acid (51.55%) and showed least preference for gallic acid (5.78%), pyrogallol (5.36%) and (below 1%) activity for L, DL tyrosine where as with resorcinol no activity was detected. Negligble activity (below 1%) of PPO activity towards tyrosine (the monophenols), suggesting the absence of monophenolase (cresolase) activity. Some authors suggest that the most common substartes for PPO are catechol, 4 methylcatechol, chlorogenic acid and catechins. Figure 6.1: Substrate specificity of A. paeoniifolius PPO Phenolic compounds (50 Mm) was used as substrate (viz. catechol, gallic acid, L tyrosine, DL tyrosine, pyrogallol, chlorogenic acid, resorcinol) to study the enzyme preference. Catechol was the most preferred substrate and its enzyme units were considered as 100%. 89

6 6.3.2 Kinetic characteristics of PPO Michaelis Menten curve of mppo activity towards increasing concentrations of substrate (catechol) is depicted in Figure 6.2. Km value reflects of the affinity of the enzyme towards substrate, smaller values shows greater affinity for its substrate. Figure 6.2: The Lineweaver Burk plot of enzyme activity measured using catechol as substrate. Km and Vmax values are mm and U/ml/min. While, Vmax is rate constant for the formation of product from the enzyme substrate (ES) complex. The K m and V max values of PPO calculated from Lineweaver Burk plot (y = x , r² = ) were found to be mm and U/ml/min, respectively. This K m value for catechol was comparable to that of tea leaf 12.5 mm [188], Mulberry Mm [189], Banana PPO of 18 mm [190], persimmon fruit 12.4 mm [191] Temperature stability The effect of temperature on PPO stability was investigated by incubating the enzyme at higher temperatures in the range of C for 5 40 min. The enzyme retained 5.68% of activity when incubated for 40 min at 75 C this was in relation to activity found at 25 C which was considered to be 100 % (Figure 6.3). 90

7 25 % Residual enzyme activity degree 65 degree 75 degree Time(min) Figure 6.3: Thermal inactivation of A. paeoniifolius PPO; Residual activity was determined after incubation at 55, 65 and 75 0 C till 5-40 min The semi-log plots were linear at all the three temperatures studied, which is consistent with inactivation by means of a simple first-order monophasic kinetics process. The inactivation rate constants (k) were calculated from the slopes of these lines (Figure 6.4). It was observed that the rate of enzyme inactivation (k) increases with increase in temperature. Higher rate constant means the enzyme is less thermostable at higher temperature [192]. k values for 55 C was lowest and at 75 C it was maximum i.e and respectively (see Table 6.1) ln (At/A0) R² = R² = degree 65 degree 75 degree -3 Time (Min) R² = Figure 6.4: Thermal inactivation kinetics of A. paeoniifolius at different temperatures 91

8 Table 6.1: Kinetics parameters of thermal inactivation of A. paeoniifolius PPO Temp( 0 C) k(min -1 ) R 2 t 1/2 (min) D(min) Z( C)=34.01 The decimal reduction time (D value) is a parameter commonly used in the estimation of enzyme stability and is defined as the time needed for 90% reduction of the initial activity. D values for A. paeoniifolius PPO ranged between min. The decimal reduction D value, at different temperatures and Z value for temperature (55-75 C) is presented in Table 6.1. Stability of enzyme enhances with increase of D value. We report the maximum D value reaches at 55 C (418.65) min and the minimum D value was at 75 C (108.10) min. The Z value is characterised by the temperature dependence of the decimal enzyme reduction activity time, which is also defined as the range of temperature increase needed for a log 10 reduction in the D value. In general, low Z values indicate greater enzyme sensitivity to heat, and high activation energy (Ea) values reflect a greater sensitivity of the enzyme to temperature change. Z value found in this study was C (Table 6.1). The energy of activation Ea is calculated by Arrhenius plot. The activation energy Ea for Amorphophallus PPO was Ea=64.22 kj/mol (y = x , r² = ).(Figure 6.5) Some of the reported Ea values for PPO include 37.9 kj/mol for Marula fruit [193] 18.6 kj/mol for banana [194], 87.8 kj/ mol for taro PPO [195]. We can state that A. paeoniifolius PPO is not very sensitive to heat and also not showing sensitivity to temperature changes. 92

9 ln k y = x R² = /T(K) Figure 6.5: Arrhenius plot of inactivation rates of A. paeoniifolius PPO enzyme. The regression equation was determined as y = x (R2 = ) Thermodynamic analysis An investigation of other thermodynamics parameters ( ΔH#, ΔS#, ΔG# and activation energy Ea) is necessary to understand further to the deactivation study and the behaviour of molecules in different physiological conditions. There is no information on the thermodynamic parameters for A. paeoniifolius PPO enzyme. Thus, the enthalpy of activation (ΔH#), Gibbs free energy (ΔG#) and entropy of activation (ΔS#) for PPO were calculated (Table 6.2). The numerical values of thermodynamic properties are affected by two major factors, namely solvent effect due to presence of surrounding water molecules and structural effect due to conformational changes occurring in the enzyme molecule during reaction. The results obtained have portrayed that enthalpy has increased in the range of kj mol 1 with increase in temperature from C. The Gibbs free energy ( G#) measures the spontaneity of a reaction. The Gibb s free energy increases with rise in temperature, with its maximum value at 75 C i.e kj mol 1 presenting that the enzyme has exhibited the resistance against thermal unfolding at higher temperatures. At all studied temperatures, entropy changes were found to be negative. The entropy of the system was found to be J mol 1 K 1 at 75 C. The possible reason for negative entropy could be formation of charged particles due to compaction of the enzyme around the enzyme molecules, associated 93

10 gains and the ordering of solvent molecules suggesting negligible disorderliness indicating that PPO enzyme from A. paeoniifolius is thermodynamically much stable [196],[197]. ΔH # and ΔS # parameters also provide a measure of the number of non covalent bonds broken and the net enzyme/solvent disorder changes related with the formation of the transition state [198]. Table 6.2: Thermodynamic parameters of heat inactivation of A. paeoniifolius PPO Ea (KJ/mol) T(K) ΔH # (KJ/mol) ΔG # (KJ/mol) ΔS # (J/mol K) CONCLUSIONS In the present chapter, the enzyme kinetics and thermal deactivation and thermodynamic properties of the PPO enzyme from A. paeoniifolius have been elucidated to expand a new insight into the structural and functional relationship of the enzyme for its stability towards temperature. It was shown that Amorphophallus corm PPO has maximum substrate specificity for catechol. Enzyme remains only 5.67% residual activity when treated at 75 0 C for 40 minutes. A. paeoniifolius PPO is very stable and not sensitive to heat and also not showing sensitivity to temperature changes and D ( min), Z(34.01 C), k ( ).The half-life time of PPO is between to min. Estimation of these (Ea, ΔG #,ΔH #,Δ S # ) thermodynamic parameters helps to understand the complex process of deactivation to some extent. Previous chapter shows that A. paeoniifolius PPO enzyme will find application in dough rising and baking industry where bond strengthening is required, this property of enzyme stability at high temperature would be economical. However detrimental effect of desired optimized heat treatment on the nutritional values the food product prepared would need to be investigated. 94