Evaluation of Inactivation Kinetics and Thermodynamic Properties of Peroxidase from Cucumis sativus for Biotechnological and Industrial Applications
Iweha Christian Chukwunyere
Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria.
Ugwoke Faith Ifeanyi
Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria and Uburu Bioresources Development Center, National Biotechnology Development Agency, Abuja, Nigeria.
Ogara Lydia Amaechi
Department of Science Laboratory Technology, Faculty of Physical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria.
Nweze Justus Amuche
Department of Science Laboratory Technology, Faculty of Physical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria.
Ogbonna Kenneth Ejike
Department of Science Laboratory Technology, Faculty of Physical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria.
Chinedum Patricia Eluka
Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria.
Akor Joseph
*
Department of Science Laboratory Technology, Faculty of Physical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria.
Odu Maureen Nwamaka
Department of Biochemistry, School of Biological Sciences, Federal University of Technology, Minna, Niger State, Nigeria.
Joshua Parker Elijah
Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria.
*Author to whom correspondence should be addressed.
Abstract
The unpurified enzyme gave specific activity of 51.36 μ/mg and thereafter it was subjected to two phases of purification process of salt precipitation and gel filtration. The precipitating agent was ammonium sulphate while Sephadex-G100 served as purification matrix. The purification fold achieved after precipitation and filtration was 3.3 and with corresponding specific activities of 34.22 μ/mg and 116.31 μ/mg. The substrate used for the assay was o-dianisidine. Within 40-80°C of temperature, the kinetics of the peroxidase inactivation was evaluated. The results from assays showed that cucumber peroxidase conformed to the hypothesis of Michealis-Menten Theory. From the Lineweaver-Burk plot, Michaelis Constant (Km) and maximum velocity (Vmax) were evaluated and obtained 5.02mg/ml and 11.57μmol/min respectively. The heat induced inactivation gave biphasic curves, where initial rise in temperature was rapidly succeeded with much slower decrease. A first-order kinetic behaviour was observed for cucumber peroxidase heat inactivation. The k values of between 3.49×10-2 to 8.38×10-2 min-1 was obtained while the Z value was found to be 22.3°C. Decrease in k values with rise in temperature suggests that cucumber peroxidase was rapidly inactivated at elevated temperature. The slope of Arrhenius plot gave the activation energy of 127.99KJMol-1K-1. Also evaluated were thermodynamic constants (ΔH, G Δ, ΔS) for inactivation of peroxidase at variable temperatures. Cucumber peroxidase activity was observed to be pH sensitive and stable within pH range of 5.6-8. Further decrease or increase from this range resulted to decrease in peroxidase stability.
Keywords: Concentration, enzyme, peroxidase, cucumber, homogenization, industrial, bioremediation